Methods for production of platelets from pluripotent stem cells and compositions thereof

ABSTRACT

Methods for production of platelets from pluripotent stem cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are provided. These methods may be performed without forming embryoid bodies or clusters of pluripotent stem cells, and may be performed without the use of stromal inducer cells. Additionally, the yield and/or purity can be greater than has been reported for prior methods of producing platelets from pluripotent stem cells. Also provided are compositions and pharmaceutical preparations comprising platelets, preferably produced from pluripotent stem cells.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/677,770 filedAug. 15, 2017, which is a continuation of U.S. Ser. No. 14/138,008 filedDec. 21, 2013, which claims the benefit of U.S. Provisional ApplicationNo. 61/787,476 filed Mar. 15, 2013 and U.S. Provisional Application No.61/740,699 filed Dec. 21, 2012, the contents of each of which areincorporated by reference herein in their entirety.

BACKGROUND OF INVENTION

Platelets are tiny blood cells that perform the vital and highlyspecialized function of blood clotting. Almost a trillion plateletscirculate in the average person's blood, and the turnover is such thatthe entire platelet population is replaced every 10 days. Thisrepresents a tremendous amount of ongoing platelet production. Plateletshave a highly organized cytoskeleton and intracellular stores of over300 proteins, which they secrete at sites of blood vessel injury.Platelets also play a role in inflammation, blood vessel growth, andtumor metastasis.

After vascular injury, platelets rapidly adhere to damaged blood vesselsand trigger a complex cascade of events that result in thrombusformation. The demand for platelet transfusions has continued toincrease during the last several decades (51). Using conventionalmethods, platelets can only be stored for less than a week, creating acontinuous challenge for donor-dependent programs. Shortages in thesupply of platelets can have potentially life-threatening consequences,especially in patients where multiple transfusions are necessary.Repeated transfusions may also lead to refractory responses that arelinked to immunity mediated host reaction and may require costly patientmatching (52; 53). The ability to generate platelets in vitro,particularly patient-matched platelets, would provide significantadvantages in these clinical scenarios.

Limitations in the supply of platelets can have potentiallylife-threatening consequences for transfusion-dependent patients withunusual/rare blood types, particularly those who are alloimmunized, andpatients with cancer or leukemia who, as often happens, develop plateletalloimmunity. Frequent transfusion of platelets is clinically necessaryin these patients because the half-life of transfused human platelets is4-5 days. Moreover, platelets from volunteer donor programs are at theconstant risk of contaminations by various pathogens. Platelets cannotbe stored frozen using conventional techniques, thus the ability togenerate platelets in vitro would provide significant advances forplatelet replacement therapy in clinical settings.

For more than a decade, human hematopoietic stem cells (HSC, CD34+) frombone marrow (BM), cord blood (CB) or peripheral blood (PB) have beenstudied for megakaryocyte (MK) and platelet generation. Using certaincombinations of cytokines, growth factors and/or stromal feeder cells,functional platelets have been produced from HSCs with significantsuccess (1; 2). However, HSCs are still collected from donors and havelimited expansion capacity under current culture conditions, whichinterferes with large-scale production and future clinical applications.

Human embryonic stem cells (hESC) can be propagated and expanded invitro indefinitely, providing a potentially inexhaustible and donorlesssource of cells for human therapy. Differentiation of hESCs intohematopoietic cells in vitro has been extensively investigated for thepast decade. The directed hematopoietic differentiation of hESCs hasbeen successfully achieved in vitro by means of two different types ofculture systems. One of these employs co-cultures of hESCs with stromalfeeder cells, in serum-containing medium (3; 4). The second type ofprocedure employs suspension culture conditions in ultra-low cellbinding plates, in the presence of cytokines with/without serum (5-7);its endpoint is the formation of cell aggregates or embryoid bodies(“EBs”). Hematopoietic precursors as well as mature, functionalprogenies representing erythroid, myeloid, macrophage, megakaryocyticand lymphoid lineages have been identified in both of the abovedifferentiating hESC culture systems (3-6:8-14). Previous studies alsogenerated megakaryocytes/platelets from hESCs by co-culturing withstromal cells in the presence of serum (15; 16). However, the yield ofmegakaryocytes/platelets in the above-described studies was low (15;16).

SUMMARY OF INVENTION

The present disclosure provides methods for production of platelets frompluripotent stem cells, such as human embryonic stem cells (hESCs) andinduced pluripotent stem cells (iPSCs or iPS cells), such as humaninduced pluripotent stem cells (hiPSCs or hiPS cells). These methods maybe performed without forming embryoid bodies, and may be performedwithout the use of stromal inducer cells. Additionally, the yield and/orpurity can be greater than has been reported for prior methods ofproducing platelets from pluripotent stem cells. Because platelets maybe produced with greater efficiency and on a larger scale, the methodsand compositions of the present disclosure have great potential for usein medicinal transfusion purposes. Additionally, because platelets donot have a nucleus and contain only minimal genetic material,preparations of the present disclosure may be irradiated beforetransfusion to effectively eliminate any contaminating nucleated cells,such as an undifferentiated hESC. Therefore, possible presence ofnucleated cells should not present a safety issue.

Platelets collected from donors have very limited shelf life and areincreasingly needed for prophylactic transfusions in patients. Incontrast to donor dependent cord blood or bone marrow CD34+ humanhematopoietic stem cells, human embryonic stem cells (hESCs) can be apromising alternative source for continuous in vitro production ofplatelets under controlled conditions. As further described herein, thedisclosure provides systems and methods to generate megakaryocytes (MKs)from pluripotent stem cells under serum- and stromal-free conditions. Inexemplary embodiments, pluripotent stem cells are directed towardsmegakaryocytes through differentiation of hemogenic endothelial cells(PVE-HE, which are further described below). A transient multi-potentialcell population expressing CD31, CD144, and CD105 markers has beenidentified at the end of PVE-HE culture. In the presence of TPO, SCF andother cytokines in feeder-free and serum-free suspension culture, up to100 fold expansion can be achieved from hESCs or hiPS cells to MKs in18-20 days. Such methods can provide robust in vitro generation of MKsfrom pluripotent stem cells. When cultured under feeder-free conditions,pluripotent stem cell-derived MKs may be used to generate platelet-likeparticles (platelet or platelet-like particle produced from humaninduced pluripotent stem cells (hiPSC-PLTs) or from human embryonic stemcells (ES PLTs)). These hiPSC-PLTs and ES-PLTs are responsive tothrombin stimulation and able to participate in micro-aggregateformation.

In one aspect, the disclosure provides a pharmaceutical preparation thatis suitable for use in a human patient comprising at least 10⁸platelets.

Additionally, the disclosure provides a pharmaceutical preparationcomprising platelets differentiated from human stem cells, e.g., atleast 10⁸ platelets. Optionally, the preparation may be substantiallyfree of leukocytes. Optionally, substantially all of the platelets maybe functional.

The pharmaceutical preparation may comprise 10⁹-10¹⁴ platelets,optionally 10⁹, 10¹⁰, 10 ¹¹, 10 ¹², 10 ¹³ or 10¹⁴ platelets.

The platelets may have one or more of the following attributes: a meanplatelet volume range of 9.7-12.8 fL; a unimodal distribution of size inthe preparation; and/or a lognormal platelet volume distribution whereinone standard deviation is less than 2 μm³ (preferably less than 1.5 μm³,1 μm³ or even 0.5 μm³).

The platelets may be positive for at least one of the following markers:CD41a and CD42b.

The platelets may be human platelets.

At least 50%, 60%, 70%, 80% or 90% of the platelets may be functional,and optionally may be functional for at least 2, 3 or 4 days afterstorage at room temperature.

In another aspect, the disclosure provides a bioreactor having weaklyadherent or non-adherent megakaryocytes that produce functionalplatelets without feeder cells.

In another aspect, the disclosure provides a composition comprising atleast 10 megakaryocyte lineage specific progenitors (MLPs).

In another aspect, the disclosure provides a cryopreserved compositioncomprising MLPs.

In another aspect, the disclosure provides a bank comprisingcryopreserved MLPs.

The MLPs may be of defined HLA types.

The cryopreserved composition may be HLA matched to a patient.

In another aspect, the disclosure provides a cryopreserved compositionor bank comprising 10⁹ to 10¹⁴ MLPs, optionally 10⁹, 10 ¹⁰, 10 ¹¹, 10¹², 10 ¹⁴ or 10¹⁴ MLPs.

In another aspect, the disclosure provides a method for producingplatelets from megakaryocytes or MPLs comprising the steps of: (a)providing a non-adherent culture of megakaryocytes; (b) contacting themegakaryocytes or MPLs with TPO or a TPO agonist to cause the formationof proplatelets in culture, wherein the proplatelets release platelets;and (c) isolating the platelets.

In another aspect, the disclosure provides a method for producingplatelets from megakaryocytes or MPLs comprising the steps of: (a)providing a non-adherent culture of megakaryocytes or MPLs; (b)contacting the megakaryocytes or MPLs with hematopoietic expansionmedium and optionally (1) TPO or a TPO agonist, SCF, IL-6 and IL-9 or(2) TPO or a TPO agonist, SCF, and IL-11, which may cause the formationof pro-platelets in culture, wherein the pro-platelets releaseplatelets. Said method may further comprise (c) isolating the platelets.

The TPO agonist comprises one or more of: ADP, epinephrine, thrombin,collagen, TPO-R agonists, TPO mimetics, second-generation thrombopoieticagents, romiplostim, eltrombopag (SB497115, Promacta), recombinant humanthrombopoietin (TPO), pegylated recombinant human megakaryocyte growthand development factor (PEG-rHuMGDF), Fab 59, AMG 531, Peg-TPOmp, TPOnonpeptide mimetics, AKR-501, monoclonal TPO agonist antibodies,polyclonal TPO agonist antibodies, TPO minibodies, VB22B sc(Fv)2, domainsubclass-converted TPO agonist antibodies, MA01G4G344, recombinant humanthrombopoietins, recombinant TPO fusion proteins, or TPO nonpeptidemimetics.

Optionally, substantially all the isolated platelets may be functional.

The non-adherent culture of megakaryocytes or MPLs may be a feeder-freeculture.

The culture in step (b) may be in a medium comprising one or more of:Stem Cell Factor (SCF) at 0.5-100 ng/ml, Thrombopoietin (TPO) at 10-100ng/ml, and Interleukin-11 (IL-11) at 10-100 ng/ml, at least one ROCKinhibitor, and/or Heparin at 2.5-25 Units/ml.

The culture in step (b) may be in a medium comprising one or more of:TPO at 10-100 ng/ml, SCF at 0.5-100 ng/ml, IL-6 at 5-25 ng/ml, IL-9 at5-25 ng/ml, at least one ROCK inhibitor, and/or Heparin at 2.5-25units/ml.

The at least one ROCK inhibitor may comprise Y27632, which Y27632 may bein a concentration of 2-20 μM, about 3-10 μM, about 4-6 μM or about 5μM.

The method may further comprise subjecting the megakaryocytes to ashearing force.

At least 2, 3, 4, or 5 platelets per megakaryocyte may be produced.

At least 50 platelets per megakaryocyte may be produced.

At least 100, 500, 1000, 2000, 5000, or 10000 platelets permegakaryocyte may be produced.

At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, orat least 95% of said platelets may be CD41a+ and/or CD42b+, e.g., CD41a+and CD42b+.

The platelets may be produced in the absence of feeder cells, and/or maybe produced in the absence of stromal feeder cells.

The platelets may be produced in the absence of any xenogeneic cells.

The platelets may be human.

The megakaryocytes or MLPs may be generated by steps comprising. (a)culturing pluripotent stem cells to form hemogenic endothelial cells(PVE-HE); (b) culturing the hemogenic endothelial cells to form MLPs;and optionally (c) culturing the MLPs to form megakaryocytes. Thepluripotent stem cells may be human.

The hemogenic endothelial cells may be derived without embryoid bodyformation.

The pluripotent stem cells may be induced pluripotent stem cells (iPSC).The iPSC may be human.

The hemogenic endothelial cells may be derived without embryoid bodyformation.

The hemogenic endothelial cells may be differentiated from thepluripotent stem cells under low oxygen conditions comprising 1% to 10%oxygen, 2% to 8% oxygen, 3% to 7% oxygen, 4% to 6% oxygen, or about 5%oxygen.

The megakaryocytes may be differentiated from the MLPs at a temperaturebetween 38-40 degrees C., or about 39 degrees C.

In another aspect, the disclosure provides a pharmaceutical preparationcomprising platelets produced by any of the method described herein,e.g., any of the methods above.

The preparation may be suitable for use in a human patient. For examplethe preparation may be suitable for use in a human patient andsubstantially free of leukocytes. Said preparation may comprise at least10⁸ platelets.

In a further aspect the disclosure provides the use of a compositioncomprising platelets (e.g., a composition as described herein, such asin the preceding paragraphs) or a composition comprising plateletsproduced by a method as described herein (e.g., a method described inthe preceding paragraphs) in the manufacture of a medicament for thetreatment of a patient in need thereof or suffering from a disease ordisorder affecting clotting or a disease or disorder treatable thereby.

The disease or disorder may comprise thrombocytopenia, trauma, ablood-borne parasite, or malaria.

In another aspect, the disclosure provides a method of treating apatient in need of platelet transfusion, comprising administering acomposition comprising platelets (e.g., a composition as describedherein, such as in the preceding paragraphs) or a composition comprisingplatelets produced by a method as described herein (e.g., a methoddescribed in the preceding paragraphs) to said patient.

The method may be effective to treat a disease or disorder comprisingthrombocytopenia, trauma, a blood-borne parasite, or malaria.

In another aspect, the present disclosure provides a compositioncomprising an isolated PVE-HE cell, which is optionally derived from apluripotent stem cell, and which is optionally produced according to themethods described herein.

In one aspect, the present disclosure provides a composition comprisingan isolated iPS-PVE-HE cell, which is optionally produced according tothe methods described herein.

In one aspect, the present disclosure provides a composition comprisingan isolated hES-PVE-HE, which is optionally produced according to themethods described herein.

In one aspect, the present disclosure provides a composition comprisingan isolated PVE-HE-MLP which is optionally derived from a pluripotentstem cell and which is optionally produced according to the methodsdescribed herein.

In one aspect, the present disclosure provides a composition comprisingan isolated iPS-PVE-HE-MLP, which is optionally produced according tothe methods described herein.

In one aspect, the present disclosure provides a composition comprisingan isolated hES-PVE-HE-MLP, which is optionally produced according tothe methods described herein.

In one aspect, the present disclosure provides a composition comprisingan isolated PVE-HE-MLP-MK which is optionally derived from a pluripotentstem cell, and which is optionally produced according to the methodsdescribed herein.

In one aspect, the present disclosure provides a composition comprisingan isolated iPS-PVE-HE-MLP-MK, which is optionally produced according tothe methods described herein.

In one aspect, the present disclosure provides a composition comprisingan isolated hES-PVE-HE-MLP-MK, which is optionally produced according tothe methods described herein.

In another aspect, the disclosure provides a pharmaceutical preparationthat is suitable for use in a human patient comprising at least 10⁸platelets, wherein the preparation is substantially free of leukocytesand wherein substantially all of the platelets are functional.

In various embodiments, the pharmaceutical preparations are irradiatedin order to remove or inactivate nucleated cells.

In another aspect, the disclosure provides a pharmaceutical preparationthat is suitable for use in a human patient comprising at least 10⁸functional platelets, wherein the mean plasma half-life of thefunctional platelets in the preparation is at least four days.

The pharmaceutical preparation of may comprise 10⁹-10¹⁴ platelets,optionally 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ or 10¹⁴ platelets.

The pharmaceutical preparation may comprise platelets having one or moreof the following attributes: a mean platelet volume range of 9.7-12.8fL; a unimodal distribution of size in the preparation; and/or alognormal platelet volume distribution wherein one standard deviation isless than 2 μm³ (preferably less than 1.5 μm³, 1 μm³ or even 0.5 μm).

The pharmaceutical preparation may comprise platelets that are positivefor at least one of the following markers: CD41a and CD41b.

At least half of the platelets may be functional for at least two,three, four or five days after storage at room temperature e.g. (22-25°C.). For example, at least 60%, 70%, 80% or 90% may be functional for atleast two days. The platelets may be stored at room temperature for atleast five days.

In another aspect, the disclosure provides a cryopreserved bank orpreparation of MLPs.

MLPs may be collected from the PVE-HE when they start to float up insuspension. Preferably the MLPs are not plated and preferably the MLPsare not allowed to adhere, thereby avoiding differentiation into othercell types and facilitating production of MKs.

The bank or preparation may comprise 10⁹ to 10¹⁴ MLPs, optionally 10⁹,10¹⁰, 10¹¹, 10¹², 10¹³ or 10¹⁴ MLPs.

One MLP may yield at least 2, 3, 4, 5, or more platelets. In exemplaryembodiments, a composition of 6×10¹⁰ to 1.2×10¹¹ MLPs is providedsufficient to generate a therapeutic dose of 300-600×10⁹ platelets at ayield of 5 platelets per MLP. In exemplary embodiments, a composition of3-6×10⁹ MLPs is provided sufficient to generate a therapeutic dose at ayield of at least 100 platelets per MLP.

In another aspect, the disclosure provides a bioreactor having weaklyadherent or non-adherent megakaryocytes that produce functionalplatelets without feeder cells. Weakly adherent cells, including saidmegakaryocytes, can be mechanically separated from each other or from asurface, for example, by washing with mild force using a serologicalpipette. Preferably the MKs are cultured under non-adherent conditions,which is thought to promote maintenance of MK phenotypes. Shear forcesmay be applied to the MK culture to improve the efficiency of plateletproduction. For example, a microfluidic chamber or chip may be used tocontrol shear stress, which may increase the platelet yield per MK. Inone aspect, MKs can be seeded into one channel of a microfluidic chipand media can be flowed past the MKs at near physiologic rates.

In another aspect, the disclosure provides a composition comprising atleast 10⁹ MLPs

In another aspect, the disclosure provides a cryopreserved compositioncomprising MLPs.

In another aspect, the disclosure provides a method for producingplatelets from megakaryocytes comprising the steps of: a) providing anon-adherent culture of megakaryocytes; b) contacting the megakaryocyteswith TPO or TPO agonists to cause the formation of proplatelets inculture, wherein the proplatelets release platelets; and c) isolatingthe platelets.

Thrombopoietin (TPO) is thought to be a key cytokine involved inthrombopoiesis and megakaryopoiesis, and is the endogenous ligand forthe thrombopoietin receptor that is expressed on the surface ofplatelets, megakaryocytes, and megakaryocytic precursors. TPO is a332-amino acid (95 kDa) glycoprotein that contains 2 domains: areceptor-binding domain (residues 1-153) and a carbohydrate-rich domain(residues 154-332) that is highly glycosylated and is important forprotein stability. The TPO receptor, c-Mpl (also known as CD110), is atypical hematopoietic cytokine receptor and contains 2 cytokine receptorhomology modules. TPO binds only the distal cytokine receptor homologymodule and thereby initiates signal transduction. In the absence of thedistal cytokine receptor homology module, c-Mpl becomes active,suggesting that the distal cytokine receptor homology module functionsas an inhibitor of c-Mpl until it is bound by TPO. Binding by TPOactivates Janus kinase 2 (Jak2) signal transducers and activators oftranscription (STAT) signaling pathway to drive cell proliferation anddifferentiation. Megakaryocyte growth and development factor (MGDF) isanother thrombopoietic growth factor. Recombinant forms of TPO and MGDF,including human and pegylated forms, may be used to induce megakaryocyteand platelet differentiation and maturation. TPO receptor-activatingpeptides and fusion proteins (i.e. Fab 59, romiplostim/AMG 531, orpegylated (Peg-TPOmp)) may be used in place of TPO. Nonpeptide mimetics(Eltrombopag (SB497115, Promacta), and AKR-501) bind and activate theTPO receptor by a mechanism different from TPO and may have an additiveeffect to TPO. TPO agonist antibodies (i.e. MA01G4G344) or minibodies(i.e. VB22B sc(Fv) 2) that activate the TPO receptor may also be used tomimic the effect of TPO. Exemplary TPO agonists are disclosed in Stasiet al., Blood Reviews 24 (2010) 179-190, and Kuter, Blood. 2007;109:4607-4616 which is each hereby incorporated by reference in itsentirety. Exemplary TPO agonists include: ADP, epinephrine, thrombin andcollagen, and other compounds identified in the literature as TPO-Ragonists or TPO mimetics, second-generation thrombopoietic agents,Romiplostim, Eltrombopag (SB497115, Promacta), first-generationthrombopoietic growth factors, recombinant human thrombopoietin (TPO),pegylated recombinant human megakaryocyte growth and development factor(PEG-rHuMGDF), TPO peptide mimetics, TPO receptor-activating peptidesinserted into complementarity-determining regions of Fab (Fab 59), AMG531 (a “peptibody” composed of 2 disulphide-bonded human IgG1-HCconstant regions (an Fc fragment) each of which is covalently bound atresidue 228 with 2 identical peptide sequences linked via polyglycine),Peg-TPOmp (a pegylated TPO peptide agonist), orally available TPOagonists, TPO nonpeptide mimetics, AKR-501, monoclonal TPO agonistantibodies, polyclonal TPO agonist antibodies, TPO minibodies such asVB22B sc(Fv)2, domain subclass-converted TPO agonist antibodies such asMA01G4G344, Recombinant human thrombopoietins, or Recombinant TPO fusionproteins, TPO nonpeptide mimetics. Wherever TPO is used as an embodimentof the invention, exemplary TPO agonists can be substituted for TPO infurther embodiments of the invention.

In another aspect, the disclosure provides a method for producingplatelets from megakaryocytes comprising the steps of a) providing anon-adherent culture of megakaryocytes or megakaryocyte progenitors, b)contacting the megakaryocytes or megakaryocyte progenitors with acomposition comprising hematopoietic expansion media to cause theformation of proplatelets in culture, wherein the proplatelets releaseplatelets, and c) isolating the platelets.

In exemplary embodiments, substantially all the isolated platelets arefunctional. In exemplary embodiments, the non-adherent culture ofmegakaryocytes or megakaryocyte progenitors is a feeder-free cultureand/or is free of xenogeneic cells. Accordingly, the disclosure providesmethods for generating platelets without feeder cells.

The culture in step (b) may be performed in a medium comprising one ormore of Stem Cell Factor (SCF), Thrombopoietin (TPO), Interleukin-11(IL-11), a ROCK Inhibitor such as Y27632 and/or Heparin. The culture instep (b) may be in a medium comprising one or more of: TPO, SCF, IL-6,IL-9, a ROCK Inhibitor such as Y27632, and/or Heparin.

In one embodiment, the hematopoietic expansion medium comprisesStemSpam™ ACF (ACF) (available from StemCell Technologies Inc.), and mayfurther comprise TPO (thrombopoietin) or TPO agonist, SCF (Stem CellFactor), IL-6 (interleukin 6) and IL-9 (interleukin 9), which may beprovided as StemSpam™ CC220 cytokine cocktail (CC220) (available fromStemCell Technologies Inc.). It may optionally comprise a ROCK inhibitorand/or Heparin. TPO, SCF, IL-6, IL-9, and IL-11 are known megakaryocytedevelopment and maturation factors (Stasi et al., Blood Reviews 24(2010) 179-190).

In one embodiment, the hematopoietic expansion medium comprisesStemline-II Hematopoietic Stem Cell Expansion Medium(Stemline-II)(available from Sigma Aldrich), and may further compriseTPO or TPO agonist, SCF, and IL-11. It may optionally comprise a ROCKinhibitor and/or Heparin. The ROCK inhibitor may be but is not limitedto Y27632.

In another embodiment, the hematopoietic expansion medium comprisesIscove's Modified Dulbecco's Medium (IMDM) as a basal medium, humanserum albumin (recombinant or purified), iron-saturated transferrin,insulin, b-mercaptoethanol, soluble low-density lipoprotein (LDL), andcholesterol (which may be referred to herein as a defined componentmedium), and may further comprise TPO or TPO agonist, SCF, and IL-11. Itmay optionally comprise a ROCK inhibitor and/or Heparin. The ROCKinhibitor may be but is not limited to Y27632.

In another embodiment, the hematopoietic expansion medium comprisesIscove's Modified Dulbecco's Medium (IMDM) as a basal medium, humanserum albumin (recombinant or purified), iron-saturated transferrin,insulin, b-mercaptoethanol, soluble low-density lipoprotein (LDL), andcholesterol (which may be referred to herein as a defined componentmedium), and may further comprise TPO (thrombopoietin) or TPO agonist,SCF (Stem Cell Factor), IL-6 (interleukin 6) and IL-9 (interleukin 9).It may optionally comprise a ROCK inhibitor and/or Heparin.

The culture in step (b) may be performed in a medium comprising ACF,Stemline-II or the defined component medium of the preceding paragraph,as well as (1) one or more of SCF (e.g., at 0.5-100 ng/ml), TPO (e.g.,at 10-100 ng/ml), IL-6 (e.g., at 5-25 ng/ml), IL-9 (e.g., at 5-25 ng/ml)and Heparin (e.g., at 2.5-25 Units/ml); (2) one or more of TPO (e.g., at10-100 ng/ml), SCF (e.g., at 0.5-100 ng/ml), IL-6 (e.g., at 5-25 ng/ml),IL-9 (e.g., at 5-25 ng/ml), Y27632 (e.g., at 5 μM, or optionally 2-20μM, or optionally an effective concentration of another ROCK inhibitor),and Heparin (e.g., at 0.5-25 units/ml); (3) one or more of TPO (e.g., at10-100 ng/ml), SCF (e.g., at 0.5-100 ng/ml), IL-11 (e.g., at 5-25ng/ml), Y27632 (e.g., at 5 μM or optionally 2-20 μM, or optionally aneffective concentration of another ROCK inhibitor), and Heparin (e.g.,at 2.5-25 Units/ml); or (4) one or more of TPO (e.g., at 10-100 ng/ml),SCF (e.g., at 0.5-100 ng/ml), IL-6 (e.g., at 5-25 ng/ml), IL-9 (e.g., at5-25 ng/ml), Y27632 (e.g., at 5 μM or optionally 2-20 μM, or optionallyan effective concentration of another ROCK inhibitor), and Heparin(e.g., at 2.5-25 Units/ml).

The method may further comprise subjecting the megakaryocytes to ashearing force.

The method may yield at least 2, 3, 4, or 5 platelets per megakaryocyte,at least 20, 30, 40 or 50 platelets per megakaryocyte, or at least 100,500, 1000, 2000, 5000, or 10000 platelets per megakaryocyte.

At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, orat least 95% of said platelets may be CD41a+ and CD42b+.

The platelets may be produced in the absence of feeder cells or stromalfeeder cells.

The platelets may be produced in the absence of any xenogeneic cells.

The platelets may be human.

The platelets may be CD41a+ and/or CD42b+.

In another aspect, the disclosure provides methods of producingmegakaryocyte progenitors (also referred to herein as MLPs) (such asthose utilized in a method of producing platelets or for anotherpurpose) which may be generated by the steps of (a) culturingpluripotent stem cells to form hemogenic endothelial cells (PVE-HE); and(b) culturing the hemogenic endothelial cells to form megakaryocytesprogenitors (MLPs). Step (a) may be carried out in the presence of ananimal-component free medium comprised of Iscove's Modified Dulbecco'sMedium (IMDM), human serum albumin, iron-saturated transferrin, insulin,b-mercaptoethanol, soluble low-density lipoprotein (LDL), cholesterol,bone morphogenetic protein 4 (BMP4) (e.g., at 50 ng/ml), basicfibroblast growth factor (bFGF) (e.g., at 50 ng/ml), and vascularendothelial growth factor (VEGF) (e.g., at 50 ng/ml). Step (b) may becarried out in Iscove's modified Dulbecco's medium (IMDM), Ham's F-12nutrient mixture, Albucult (rh Albumin), Polyvinylalcohol (PVA),Linoleic acid, SyntheChol (synthetic cholesterol), Monothioglycerol(a-MTG), rh Insulin-transferrin-selenium-ethanolamine solution,protein-free hybridoma mixture II (PFHMII), ascorbic acid 2 phosphate,Glutamax I (L-alanyl-L-glutamine), Penicillin/streptomycin, Stem CellFactor (SCF) at 25 ng/ml, Thrombopoietin (TPO) (e.g., at 25 ng/ml),Fms-related tyrosine kinase 3 ligand (FL) (e.g., at 25 ng/ml),Interleukin-3 (IL-3) (e.g., at 10 ng/ml), Interleukin-6 (IL-6) (e.g., at10 ng/ml), and Heparin (e.g., at 5 Units/ml).

In another aspect, the disclosure provides methods of producingmegakaryocytes (such as those utilized in a method of producingplatelets or for another purpose) which may be generated by the steps of(a) culturing pluripotent stem cells to form hemogenic endothelial cells(PVE-HE); (b) culturing the hemogenic endothelial cells to form MLPs;and (c) culturing the MLPs to form megakaryocytes as shown in Examples 1and 2. Steps (a) and (b) may be carried out as described in thepreceding paragraph. Step (c) may be carried out in Iscove's ModifiedDulbecco's Medium (IMDM) as basal medium, human serum albumin,iron-saturated transferrin, insulin, b-mercaptoethanol, solublelow-density lipoprotein (LDL), cholesterol, TPO (e.g., at 30 ng/ml), SCF(e.g., at 1 ng/ml), IL-6 (e.g., at 7.5 ng/ml), IL-9 (e.g., at 13.5ng/ml), and optionally a ROCK inhibitor such as but not limited toY27632 (e.g., at 5 μM), and/or Heparin (e.g., at 5-25 units/m).

In another aspect, the disclosure provides a pharmaceutical preparationcomprising platelets produced by the methods above. The preparation maybe suitable for use in a human patient and/or may comprise at least 10⁸platelets, and/or may be substantially free of leukocytes.

In another aspect, the disclosure provides the use of the platelets ofany composition described herein or produced by any method hereindescribed in the manufacture of a medicament for the treatment of apatient in need thereof or suffering from a disease or disorderaffecting clotting.

In another aspect the disclosure provides a method of treating a patientin need of platelet transfusion, comprising administering platelets ofany composition described herein or produced by any method hereindescribed to said patient, which may be in an amount effective to treata disease or disorder affecting clotting and/or other platelet functionand/or another disorder that may be treated thereby, such asthrombocytopenia or trauma. Thrombocytopenia results from disturbancesin platelet production, distribution, or destruction. It is frequentlyfound in a variety of medical conditions, including liver cirrhosis, HIVinfection, autoimmune disease, chemotherapy-induced myelosuppression andbone marrow disorders. A low platelet count is associated with anincreased risk of bleeding. An additional exemplary disease or disorderthat may be treated thereby is malaria and other parasitic infections,which while not intending to be limited by theory is thought to bemediated by the ability of the human platelet factor 4 to kill malariaparasites within erythrocytes by selectively lysing the parasite'sdigestive vacuole (see Love et. al., Cell Host Microbe 12 (6): 815-23,which is hereby incorporated by reference in its entirety).

In another aspect the disclosure provides a method of drug delivery,comprising administering platelets of any composition described hereinor produced by any method herein described to said patient, wherein saidplatelets deliver said drug. For example, it is thought that due totheir in vivo life span, lack of engraftment post administration, andhoming properties, platelets may be useful as a drug carrier. hESCs,hiPCs and MLPs may be genetically modified and used to produce plateletsthat express a desired drug for treatment of a disease. In one aspect,hESCs, hiPCs or MLPs could be genetically modified to express anantitumor agent. Platelets produced from such genetically modifiedhESCs, hiPSCs and MLPs may be used to deliver such antitumor agent to atumor for the treatment of a neoplastic disease.

A further aspect of the disclosure provides a method of producing a β2microglobulin-deficient platelet, e.g., a reduced immunogenicity or“universal” platelet, comprising use of any method as disclosed hereinto produce a platelet from a cell engineered to be deficient in β2microglobulin expression, such as a β2 microglobulin knockoutpluripotent cell. The disclosure also provides a β2microglobulin-deficient platelet, megakaryocyte, or platelet progenitorlacking expression of β2 microglobulin. A β2 microglobulin-deficientplatelet generally has low or preferably undetectable Class I MHCmolecules present in its plasma membrane, thereby reducingimmunogenicity of the platelet.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Stepwise process depicting the generation of platelets frompluripotent stem cells. This Figure shows the progression ofdifferentiation from pluripotent stem cells through pluripotent-derivedhemogenic endothelial cells (PVE-HE) through megakaryocytelineage-specific progenitor cells (MLP) and through megakaryocytes (MK).

FIGS. 2 A-C. Progression of differentiation of iPS cells throughadvanced differentiation morphology cells (ADM). These Figures show iPScell progression toward PVE-HE. FIG. 2A, after 48 hours attached cellsshow typical pluripotent stem cell morphology under feeder-freecondition. FIG. 2B, the almost complete transition from pluripotent stemcell morphology into scattered small cell clusters is shown. FIG. 2C,after 96 to 146 hours post PVE-HE differentiation initiation, advanceddifferentiation morphology was observed showing small compact cellclusters growing on top of the monolayer.

FIGS. 3 A-B. Characterization of advanced differentiation morphology ofPVE-HE. These Figures show the phenotype and morphology of ADM cellsthat have differentiated into PVE-HE. The ADM cells show the PVE-HEphenotype CD31⁺CD144(VE-Cad)⁺CD105⁺ at this stage of differentiation(FIG. 3A). ADM cells were also analyzed for morphologic changes (FIG.3B).

FIG. 4. Characterization of iPS-PVE-HE-derived megakaryocyticlineage-specific progenitors (MLPs) by flow cytometry. This Figure showsthe phenotype of human iPS-PVE-HE-MLP cells asCD34⁺CD31⁺CD41a⁺CD43⁺CD13⁺CD14⁻CD42b^(−/+).

FIGS. 5 A-C. Morphologic analysis of human iPS-PVE-HE-MLP-derivedmaturing MK cells. These Figures show nuclei inside MK cells (indicatedby “N”), and readily observable proplatelet forming cells with elongatedpseudopodia (indicated by arrows). At 72 hours post initiation ofplatelet differentiation, very large polyploidy MKs (50 μM) becameabundant with progression of maturation (FIGS. 5A & B. Scale bar in 5Ais 100 μM, N in 5B indicates nucleus inside MK). From between 72-96hours proplatelet forming cells with elongated pseudopodia were readilyobserved microscopically. (FIGS. 5A & 5C, indicated by arrows).

FIGS. 6 A-E. Comparison of phenotype and purity of platelet preparationsfrom different sources. These Figures show flow cytometric analysis ofthe morphology and cell surface expression of CD41a and CD42b onperipheral blood derived human platelets and iPS-PVE-HE-MLP-MK-derivedplatelets. Between 72-96 hours post initiation, the amount ofCD41a+CD42b+platelets increased dramatically, reaching levels as high asabout 70% (FIG. 6D). FIGS. 6A-6B show forward scatter (“FSC-A”) and sidescatter (“SSC-A”) for donor-derived human platelets (6A), andhES-derived platelets (hES-PVE-HE-MLP) produced as described in Example3 (6B). FIGS. 6C-E show expression of CD41a and CD42B by donor-derivedhuman platelets (6C), iPS-derived platelets (iPS-PVE-HE-MLP) (6D), andhES-derived platelets (hES-PVE-HE-MLP) (6E), with the latter two sampleshaving been produced as described in Example 3.

FIG. 7. Ultrastructural comparison of peripheral blood derived humanplatelets and platelets produced by differentiation of human inducedpluripotent cells (hiPSC-PLTs) by transmission scanning microscopy. ThisFigure shows the similarities in cellular characteristics of peripheralblood derived human platelets and hiPSC-PLTs of the present disclosure.The hiPSC-PLTs are discoid.

FIG. 8. Comparison of peripheral blood derived human platelets (hPRP)and hiPSC-PLTs. This Figure shows similarities between the two cellpreparations in platelet diameter (top panel), and expression ofstructural cell proteins beta1-tubulin and F-actin (via FITC-phalloidinbinding)(bottom panel), which are involved in activation inducedplatelet shape changes. Negative Hoechst staining (bottom panel, topright) confirmed the absence of nuclear DNA in iPSC-PLTs anddonor-derived PLTs.

FIG. 9. Comparison of peripheral blood derived human platelets andhiPSC-PLTs. This Figure shows the similarities in morphologiccharacteristics of peripheral blood derived human platelets andhiPSC-PLTs of the present disclosure using differential interferencecontrast (DIC) live-cell microscopy. Both the peripheral blood derivedhuman platelets and the hiPSC-PLT show pseudopodia emission indicativeof activation when bound to the negatively charged glass surface.

FIGS. 10 A-B. Comparison of peripheral blood derived human platelets andhiPSC-PLTs. These Figures show the similarities of alpha-granuleexpression as demonstrated by Thrombospondin 4 (TSP4) and PlateletFactor 4 (PF4) labeling. TSP4 and PF4 are chemokines released fromalpha-granules of activated platelets. hiPSC-PLTs (FIG. 10B) were seento have normal alpha-granule expression relative to normal humanplatelets (FIG. 10A), as demonstrated by TSP4 and PF4 labeling.

FIG. 11. Functional evaluation of hiPSC-PLTs. This Figure shows the invitro activation of hiPSC-PLTs with thrombin as measured by theupregulation of two adhesion molecules CD62p and αIIbβII (as measured bythe PAC-1 Ab).

FIGS. 12 A-B. Functional comparison of circulating human platelets andplatelets derived from human iPSCs and ESCs. These Figures show the invivo clot forming potential of circulating human platelets, hiPSC-PLTsand hESC-PLTs. Experiments were performed with natural human platelets(“hPLT”), iPSC-PLT, and hES cell derived platelets (“hESC-PLT”). FIG.12A shows representative images of thrombi formed in a mouse vessel wallinjury model. FIG. 12B graphically illustrates the average number ofplatelets bound to the thrombus in each experiment. Platelet binding wasinhibited by treatment with ReoPro, an anti-αIIbβIII antibody fragment,indicating that binding was dependent on αIIbβIII as expected. (FIG.12B).

FIG. 13. Kinetics of PLTs in macrophage-depleted NOD/SCID mice afterinfusion. This Figure shows a detectable circulation of hiPSC-PLTs andhESC-PLTs of eight hours.

FIGS. 14 A-E. Exemplary process flow diagrams for production ofplatelets from pluripotent stem cells.

FIG. 15. FACS Analysis, CD41a, CD42b—MLP Derived From hiPSC.

FIG. 16. Representative MLPs Derived from hiPSC.

FIG. 17. FACS Analysis CD31+, CD43+—MLP Derived from hiPSC.

FIG. 18. Representative MKs derived from hESC.

FIG. 19. Proplatelet Formation from MK.

FIG. 20. FACS Analysis CD41a, CD42b.

FIG. 21. DIC Microscopy with β1-Tubulin Staining, Human Donor PLT (top)hESC-PLT (bottom).

DETAILED DESCRIPTION OF INVENTION

Lists of definitions and abbreviations used in this disclosure areprovided at the end of the Detailed Description.

As noted above, limitations in the supply of platelets can havepotentially life-threatening consequences for transfusion-dependentpatients. Pluripotent cells can be propagated in vitro indefinitely, andrepresent a potentially inexhaustible and donorless source of plateletsfor human therapy. The ability to create banks of hESC lines withmatched or reduced incompatibility could potentially decrease oreliminate the need for immunosuppressive drugs and/or immunomodulatoryprotocols. Exemplary embodiments provide a method comprising producingpatient-specific iPS cells (for example using methods herein describedor any others known in the art), and producing patient-specificplatelets from said patient-specific iPS cells, which platelets may beused for treatment of patients, such as patients who have developed orare at risk of developing platelet alloimmunity.

Exemplary embodiments provide an efficient method to generatemegakaryocytes (MKs) from pluripotent stem cells under serum-free andfeeder-free conditions. Preferably the MKs are produced frommegakaryocyte lineage-specific progenitor cells (MLPs, which are furtherdescribed herein). Preferably the MKs are not produced fromhemangioblasts or hemangio-colony forming cells such as those disclosedin U.S. Pat. No. 8,017,393. Using methods disclosed herein, pluripotentstem cells (iPSC and ESCs) were directed towards MK differentiation. Theefficiency of differentiation of megakaryocytes from MLPs has been veryhigh (up to 90%). Without further purification, 85% of live cells fromthe MK suspension cultures were CD41a+CD42b+ and the mature MKs werealso CD29+ and CD61+. These in vitro-derived MK cells can undergoendomitosis and become mature, polyploid MKs. Importantly, proplateletforming cells with elongated pseudopodia were observed at the late stageof MK culture, indicating that MKs generated in this system are able toundergo terminal differentiation and generate functional platelets underfeeder-free conditions.

Described herein is an efficient system which is adaptable for largescale and efficient in vitro megakaryocyte production using iPSC orother pluripotent stem cells as source cells under controlledconditions. Additionally, the platelets may be produce in sufficientquantities to supplement or supplant the need for donor-derivedplatelets. Additionally, the disclosed methods may be utilized toproduce platelets and platelet progenitor cells in a predictable manner,such that the cells may be produced “on demand” or in quantities desiredto meet anticipated need. The cells expressed CD41a and CD42b, underwentendomitosis, and formed mature polyploid MKs. Upon further maturation,they generated functional or activated platelets that were stimulated bythrombin to become positive for cell adhesion molecules, CD62p andαIIbβIII (thought to occur by exposure of the PAC-1 binding site afterconformational change of αIIbβIII upon activation, while CD62p isthought to be exposed on outside membrane due to granule release). Bothof these markers are known to be expressed on the surface of activatedplatelets and were detected on the hiPSC-PLTs using a PAC-1 and CD62p(p-selectin) binding assay. Because no stromal inducer cells are neededfor megakaryocyte production, the methods described herein can providefor feeder-free platelet generation, such as platelet generation withoutany use of xenogeneic cells.

In exemplary embodiments, additional factors including estradiol,vitamin B3 and extracellular matrix proteins may also be used to enhanceplatelet production, such as by stimulating megakaryocyte maturationand/or stimulating platelet production, which may be carried out in theabsence of stromal cells.

The production of megakaryocytes under serum-free and stromal freeconditions may allow screening for factors that are critical inregulating megakaryopoiesis and thrombopoiesis under well-definedconditions. Factors so identified may contribute to clinicalapplications. Advances in this area may also likely provide insightsinto the cellular and molecular mechanisms regulating different aspectsof megakaryopoiesis including lineage commitment, expansion andmaturation.

Exemplary embodiments integrate step-wise inductions of megakaryocytedifferentiation from pluripotent stem cells. Further optimization andestablishment of in-process controls can be performed to improve theconsistency and efficiency of this system for clinical applications. Theunderlying cellular or extra-cellular mechanisms regulatingmegakaryocyte maturation need not be completely defined in order topractice these methods. Other factors that promote polyploidization andcytoplasmic maturation may be identified and included to facilitate theterminal differentiation of in vitro-derived megakaryocytes. Forinstance, at least one ROCK kinase inhibitor may be used to induceendomitosis of megakaryocytes at an early stage. However, this effect isthought to be likely due to an artificial blocking of chromosomesegregation and cytokinesis rather than an orchestrated cellular andnuclear maturation of a differentiating megakaryocyte. It may beadvantageous to reach a balance between the expansion, endomitosis andthe cytoplasmic maturation to achieve the highest in vitro megakaryocyteyields, terminal differentiation status and downstream production offunctional platelets under defined conditions.

These current results demonstrated that platelets derived frompluripotent stem cells share morphological and functional properties ofnormal blood platelets. These pluripotent stem cell derived humanplatelets are able to function in vivo as well.

Additional hemodynamic events may occur during the generation andpropagation of platelet thrombi in the living organism which may not befully mimicked by in vitro systems. The availability of intravitalimaging technology provides a means to directly examine and quantify theplatelet-dependent thrombotic process that occurs after vascular injuryin complex in vivo systems. Using intravital high-speed widefieldmicroscopy, the inventors demonstrated that pluripotent stemcell-derived platelets are incorporated into the developing mouseplatelet thrombus at the site of laser-induced arteriolar wall injury inliving mice similarly to normal human blood platelets. Pretreatment ofthe pluripotent cell-derived and control platelets with ReoPro markedlyreduced the number of both donated and pluripotent stem cell-derivedplatelets incorporating in the thrombi, confirming the binding wasmediated by αIIbβII integrin. These results indicate that pluripotentstem cell-derived platelets are functional at the site of vascularinjury in living animals.

Platelets are anucleate cells that adhere to tissue and to each other inresponse to vascular injury. This process is primarily mediated by theplatelet integrin αIIbβIII, which binds to several adhesive substratessuch as von Willebrand Factor (vWF) and fibrinogen to bridge and furtheractivate platelets in a growing thrombus (36). The results hereindemonstrate that platelets generated from pluripotent stem cells arefunctionally similar to normal blood platelets both in vitro and inliving animals. The pluripotent stem cell-derived platelets were shownto possess important functions involved in hemostasis, including theability to aggregate when stimulated with physiological agonists. Inaddition, immunofluorescence and transmission electron microscopicresults further demonstrate that platelets generated from pluripotentstem cells are similar to normal blood platelets.

As further described in the examples below, numerous similaritiesbetween pluripotent cell-derived platelets and purified normal humanplatelets were observed. These similarities include the following.

hiPSC-PLTs are discoid (as demonstrated by transmission electronmicroscopy)

hiPSC-PLTs are mostly ultrastructurally identical to circulating humanPLTs (as demonstrated by transmission electron microscopy).

The size of hiPSC-PLTs is comparable to that of circulating human PLTs(2.38 μm±0.85 μm versus 2.27 μm 0.49 μm) as demonstrated by DIC and1-tubulin IF microscopy)

hiPSC-PLTs were able to spread on glass and form both filopodia andlamelopodia as demonstrated by DIC live-cell microscopy images).

hiPSC-PLTs are anucleate—comparable to circulating human PLTs (asdemonstrated by Hoechst labeling).

hiPSC-PLTs have a normal tubulin cytoskeleton relative to circulatinghuman PLTs (as demonstrated by β1-tubulin labeling).

hiPSC-PLTs have normal filamentous actin relative to circulating humanPLTs (as demonstrated by phalloidin labeling).

hiPSC-PLTs have normal alpha-granule expression relative to circulatinghuman PLTs (as demonstrated by TSP4 and PF4 labeling).

Another scientific and clinical issue is whether pluripotent stemcell-derived platelets are functional in the complex in vivo setting. Alarge number of experimental models were established in the past decadeto investigate thrombus formation in mice, including the laser-injurythrombosis model recently used by several groups (37:38:39). Thelaser-induced thrombosis model initiates platelet thrombus formation asfast as 5-30 seconds following injury. Therefore, this model allows themonitoring of the real-time incorporation of rapidly cleared humanplatelets and hESC-PLTs into the developing mouse platelet thrombus,which involves a large number of signaling pathways, enzymatic cascades,as well as the interplay of a myriad of cellular and protein components.This model also mirrors the inflammatory reactions associated withthrombin-induced thrombosis.

Using the laser-induced vessel injury model, the intravital microscopyanalyses demonstrate that hiPSC-PLTs and hESC-PLTs, like bloodplatelets, are incorporated into the developing mouse platelet thrombusthrough αIIbβIII integrin following vascular injury (FIG. 12A).hiPSC-PLTs and hESC-PLTs functional capabilities were determined to bemediated by αIIbβIII by pretreatment with ReoPro, a Fab fragment of ahuman-murine chimeric monoclonal antibody that binds specifically toαIIbβIII and inhibits platelet function (FIG. 12B). These resultsprovide evidence that hESC-PLTs are functional at the site of vascularinjury in vivo. Importantly, it is shown for the first time thatplatelets derived from pluripotent stem cells under serum and feederfree conditions are able to facilitate coagulation and thrombusformation in vivo.

Two previous studies have reported the generation of MKs from hESCs. Theyield in these systems is extremely low, and unlike the current system,relies on co-culture with animal stromal cells supplemented with serum(15,16). Moreover, in vivo functionality was not reported (15,16). Theelimination of these two variables in pluripotent stem celldifferentiation allows the generation of platelets without exposure toanimal products. Additionally, the present disclosure demonstrates thatthe feeder-free system described herein can generate MKs with highefficiency and that functional platelets can be efficiently generatedunder the feeder-free conditions.

Thrombopoiesis is a highly complex process, with sophisticatedreorganization of membrane and microtubules and precise distributions ofgranules and organelles (40). Despite recent advances in theunderstanding of platelet biogenesis, mechanistic details underlyingmembrane reorganization, initiation of proplatelets, transportation ofplatelet organelles and secretary granules, and control of platelet sizeremain to be elucidated. The ability to generate MKs under serum- andfeeder-free conditions should aid in the screening of factors that arecritical in regulating different aspects of megakaryopoiesis underwell-defined conditions, including lineage commitment, expansion andmaturation.

This disclosure provides various methods for producing PVE-HE cells,MLPs, MK, proplatelets and platelets in vitro (or ex vivo) that areiPS-derived or ESC-derived.

This disclosure provides methods for transitioning from an iPS cell orES cell to a PVE-HE cell, or to a MLP, or to a MK, or to a platelet.This disclosure provides methods for transitioning from a PVE-HE cell toa MLP, or to a MK, or to a platelet. This disclosure provides methodsfor transitioning from a MLP to a MK, or to a platelet. This disclosureprovides methods for transitioning from a MK to a platelet. Thesevarious cultures are described briefly below.

PVE-HE cells may be produced from iPS or ES cells through a method thatcomprises culturing iPS cells or ES cells in a culture medium comprisingIscove's Modified Dulbecco's Medium (IMDM) as basal medium, human serumalbumin, iron-saturated transferrin, insulin, b-mercaptoethanol, solublelow-density lipoprotein (LDL), cholesterol, and further comprising bonemorphogenetic protein 4 (BMP4) (e.g., at 50 ng/ml), basic fibroblastgrowth factor (bFGF) (e.g., at 50 ng/ml), and vascular endothelialgrowth factor (VEGF) (e.g., at 50 ng/ml). This culture period may lastan average of 6 days.

MLPs may be produced from PVE-HE cells through a method that comprisesculturing PVE-HE cells in a culture medium comprising Iscove's modifiedDulbecco's medium (IMDM), Ham's F-12 nutrient mixture, Albucult (rhAlbumin), Polyvinylalcohol (PVA), Linoleic acid, SyntheChol (syntheticcholesterol), Monothioglycerol (a-MTG), rhInsulin-transferrin-selenium-ethanolamine solution, protein-freehybridoma mixture II (PFHMII), ascorbic acid 2 phosphate, Glutamax I(L-alanyl-L-glutamine), Penicillin/streptomycin, and further comprisingStem Cell Factor (SCF) (e.g., at 25 ng/ml), Thrombopoietin (TPO) (e.g.,at 25 ng/ml), Fms-related tyrosine kinase 3 ligand (FL) (e.g., at 25ng/ml), Interleukin-3 (IL-3) (e.g., at 10 ng/ml), Interleukin-6 (IL-6)(e.g., at 10 ng/ml), and optionally Heparin (e.g., at 5 Units/ml). Thisculture period may last an average of 3-4 days. The MLPs harvested fromthese cultures may be cryopreserved or used immediately for plateletproduction or other analysis.

Megakaryocytes may be produced from MLPs through a method that comprisesculturing the MLPs in a medium that comprises Iscove's ModifiedDulbecco's Medium (IMDM), human serum albumin, iron-saturatedtransferrin, insulin, b-mercaptoethanol, soluble low-density lipoprotein(LDL), cholesterol, and further comprises TPO (e.g., at 30 ng/ml), SCF(e.g., at 1 ng/ml), IL-6 (e.g., at 7.5 ng/ml), IL-9 (e.g., at 13.5ng/ml), and optionally a ROCK inhibitor such as Y27632 (e.g., at 5 μM),and/or Heparin (e.g., at 5-25 units/ml).

This latter culture produces MK and platelets depending on the length ofthe culture. Platelets are typically observed by about days 3-4 ofculture. It will be understood that during the culture period the MLPwill be maturing into MK, and the MK will be maturing into proplatelets,and the proplatelets will be maturing into platelets.

Platelets therefore may be produced from MLPs or MKs through a methodthat comprises culturing the MLPs or MKs in a medium that comprisesIscove's Modified Dulbecco's Medium (IMDM), human serum albumin,iron-saturated transferrin, insulin, b-mercaptoethanol, solublelow-density lipoprotein (LDL), cholesterol, and further comprises TPO(e.g., at 30 ng/ml), SCF (e.g., at 1 ng/ml), IL-6 (e.g., at 7.5 ng/ml),IL-9 (e.g., at 13.5 ng/ml), and optionally a ROCK inhibitor such asY27632 (e.g., at 5 μM), and/or Heparin (e.g., at 5-25 units/ml). Theseculture period may be 4-8 days in length, or more.

Megakaryocyte Lineage Progenitors

Various embodiments of the present disclosure provide for a method ofgenerating Megakaryocyte Lineage Progenitors (MLPs) from pluripotentstem cells (including human iPS and human ES), as well as compositionsof MLPs.

Early lineage hemogenic endothelial cells are CD41a negative, expressingCD41a during late-stage hemogenic differentiation in hematopoieticprogenitors. CD42b is expressed exclusively in mature megakaryocytes.MLP cultures may be heterogeneous with a high percentage of CD41+ cellsand a low percentage of CD42+ cells.

MLPs may be assessed for the percentage of CD41a and CD42b doublypositive cells by FACS analysis. CD41a is a subunit of fibrinogenreceptor (αIIbβIII) and CD42b is a subunit on von Willebrand Factorreceptor (GPIb-V-IX). The expression of both receptors is specific formegakaryocyte lineages and both are thought to be required for plateletfunction.

Prior to harvesting for cryopreservation MLPs may be assessed for theapproximate percentage of attached cells and the extent ofdifferentiated large cells with low nuclei to cytoplasm ratio. Attachedcells may appear as diffuse colonies with no clear colony borders. Theremay be an abundance of the floating MLPs resting on top of the attachedcell population. Viable floating MLPs may appear clear, with minimalbirefringence, demarked by a smooth cell membrane.

MLPs and compositions thereof may optionally be provided as acryopreserved composition.

Megakaryocytes

Various embodiments of the present disclosure provide for a method ofgenerating megakaryocytes from pluripotent stem cells (including humaniPS and human ES) under stromal-free conditions and/or under serum-freeconditions. These embodiments include generating megakaryocytes. Furtherembodiments provide for a method of generating megakaryocytes frompluripotent derived hemogenic endothelial cells. Said megakaryocytes arepreferably able to produce platelets, e.g., when cultured under theconditions as described herein.

In one embodiment, the method comprises: providing pluripotent stemcells; and differentiating the pluripotent stem cells intomegakaryocytes. In one embodiment, the pluripotent stem cells are humancells. In another embodiment, the pluripotent stem cells are hESCoptionally produced without the destruction of the embryo such as theNED7 line. In another embodiment, the pluripotent cells are human EScells. In another embodiment, the megakaryocytes are derived frominduced pluripotent stem cells. In another embodiment, the pluripotentstem cells are human iPS cells derived from reprogramming somatic cells.In one embodiment, the somatic cells are from fetal tissue. In anotherembodiment, the somatic cells are from adult tissue.

In another embodiment, the present disclosure provides for a method ofscreening for a modulator of cellular differentiation comprising:providing a quantity of megakaryocytes (MKs); contacting the MKs with atest compound; and determining the presence or absence of a functionaleffect from the contact between the MKs and the test compound, whereinthe presence of a functional effect indicates that the test compound isa megakaryopoietic, thrombopoietic, and/or hematopoietic factor thatmodulates cellular differentiation and the absence of a functionaleffect indicates that the test compound is not a megakaryopoietic,thrombopoietic, and/or hematopoietic factor that modulates cellulardifferentiation. In other embodiments, megakaryopoietic, thrombopoietic,and/or hematopoietic factors relate to the expansion, endomitosis,cytoplasmic maturation, and terminal differentiation of functionalplatelets.

Platelets

Other embodiments of the present disclosure provide for a method ofgenerating platelets from human embryonic stem cells and pluripotentstem cells (including iPSC and human iPSC). In one embodiment, themethod comprises: providing human embryonic stem cells (hESCs); formingPVE-HE cells differentiating the PVE-HE cells into megakaryocytes; anddifferentiating the megakaryocytes into platelets.

In another embodiment, the method of generating platelets comprises:providing PVE-HE cells, differentiating the PVE-HE cells into MLPs ormegakaryocytes; and optionally differentiating the MLPs intomegakaryocytes; and then differentiating (or maturing) themegakaryocytes into platelets, typically through a proplatelet step. Theprocess of differentiating the PVE-HE cells into megakaryocytes can beperformed as described above. In one embodiment, the PVE-HE cells arederived from human ES cells. In another embodiment, the PVE-HE cells arederived from induced pluripotent stem cells (iPSCs). In one embodiment,the iPS cells are human iPS cells derived from reprogramming somaticcells. In one embodiment, the somatic cells are from fetal tissue. Inanother embodiment, the somatic cells are from adult tissue. In variousembodiments, the process of differentiating the megakaryocytes intoplatelets comprises continuing to culture the megakaryocytes to allowthe megakaryocytes to differentiate into platelets. In variousembodiments, the process of differentiating the megakaryocytes intoplatelets is under feeder free conditions and comprises collecting themegakaryocytes differentiated from megakaryocyte-lineage specificprogenitor cells.

In further embodiments, platelets are collected from Day 4 to Day 10 ofmegakaryocyte culture in MK-M medium or other medium comprising Iscove'sModified Dulbecco's Medium (IMDM) as basal medium, human serum albumin,iron-saturated transferrin, insulin, b-mercaptoethanol, solublelow-density lipoprotein (LDL), cholesterol, TPO (e.g., at 30 ng/ml), SCF(e.g., at 1 ng/ml), IL-6 (e.g., at 7.5 ng/ml), IL-9 (e.g., at 13.5ng/ml), and optionally a ROCK inhibitor such as Y27632 (e.g., at 5 μM),and/or Heparin (e.g., at 5-25 units/ml). In a preferred embodiment,platelets are collected from 3-5 days after the first emergence ofproplatelet forming cells in the megakaryocyte culture in MK-M medium.In certain embodiments, the platelets are purified using densitygradient centrifugation. In further embodiments, the density gradientcentrifugation uses Percoll medium. In further embodiments, the densitygradient centrifugation uses BSA/HSA medium. In another embodiment, theplatelet purification method separates particles that are CD41anegative. In another embodiment, the platelet purification methodseparates particles that are CD42b negative. In another embodiment, theplatelet purification method retains cell viability and morphologicalintegrity. In other embodiments, the platelets express CD41a and CD42b.In other embodiments, the platelets are responsive to thrombinstimulation. In another embodiment, the platelets are able to spread onfibrinogen and von Willebrand Factor (vWF) surfaces. In additionalembodiments, the platelets have capacity for PAC-1 binding and integrinactivation. In another embodiment, the platelets form micro-aggregatesand facilitate clot formation and retraction. In another embodiment, theplatelets are not activated in the presence of apyrase and/or EDTA.

Various embodiments of the present disclosure provide for a method ofusing pluripotent stem cell-derived platelets. In certain embodiments,the pluripotent stem cell-derived platelets are used in platelettransfusions. The method may comprise providing a quantity ofpluripotent stem cell-derived platelets; and administering the quantityof pluripotent stem cell-derived platelets to a subject in need thereof.In various embodiments the pluripotent stem cell-derived platelets canbe patient-matched platelets. In another embodiment, the platelets arederived from iPSCs. In a certain embodiment, the platelets are derivedfrom human iPS cells. In other embodiments, the platelets are stored ina solution that does not contribute to an HLA alloimmunogenic responsein a subject upon administration of the platelets to the subject. Inadditional exemplary embodiments, the pluripotent stem cell-derivedplatelets may be substantially free of leukocytes, for examplecontaining less than 5%, less than 4%, less than 3%, less than 2%, orless than 1% leukocytes, preferably less than 0.1%, 0.001% or even0.0001%. Additional exemplary embodiments provide a preparation ofpluripotent stem cell-derived platelets which contains less than 10⁶leukocytes in the preparation, more preferably less than 10⁵, 10 ⁴, oreven 10³ leukocytes.

Additional exemplary embodiments provide a composition containing atleast 10⁸ platelets, more preferably at least 10, at least 10¹⁰, or atleast 10¹¹ platelets.

Using the current blood bank storage conditions at 22-24° C., theviability and function of human platelets (collected by aphaeresis) canbe maintained for only 5 days—the limited storage time though to be dueto aging of the platelets and an increasing risk of bacterialproliferation. It is expected that platelets produced by the methods ofthe present invention will have a longer shelf life than bankedplatelets, such as being able to be maintained for at least 6, 7, 8, 9,10, 11, 12, 13, 14 or even 15 days at 22-24° C. and maintain appropriateviability to be used in human patients.

In certain embodiments, the platelets can be treated—after isolation orduring one or more of the culturing steps leading to plateletproduction—with one or more agents that prolong platelet storage at22-24° C., refrigerated (e.g., 4° C.) and/or frozen.

For example, the present invention contemplates treating the plateletswith agents, or deriving the platelets under conditions, that reducesialidase activity and (optionally) inhibit proliferation of one or morebacteria in a platelet product preparation. The method can include thesteps of contacting the platelet product preparation with an amount of asialidase inhibitor, to thereby obtain a sialidase treated plateletproduct preparation; wherein the sialidase activity is reduced and theproliferation of one or more bacteria is inhibited, as compared to aplatelet product preparation not subjected to a sialidase inhibitor.

The type of bacteria inhibited include those commonly found in plateletproduct preparations. Examples of such bacteria include: Aspergillus,Bacillus sp, Bacteroides eggerthii, Candida albicans, Citrobacter sp,Clostridium perfringens, Corynebacterium sp, Diphtheroid, Enterobacteraerogenes, Enterobacter amnigenus, Enterobacter cloacae, Enterococcusavium, Enterococcus faecalis, Escherichia coli, Fusobacterium spp.,Granulicatella adiacens, Heliobacter pylori, Klebsiella sp, (K.pneumonia, K. oxytoca), Lactobacillus sp, Listeria sp, Micrococcus sp,Peptostreptococcus, Proteus vulgaris, Pseudomonas sp, Pseudomys oxalis,Propionibacterium sp, Salmonella sp, Serratia sp, Serratia marcescensStaphylococcus sp (Coagulase-negative Staphylococcus, Staphylococcusepidermidis, Staphylococcus aureus), Streptococcus sp, (S. gallolyticus,S. bovis, S. pyogenes, S. viridans), and Yersinia enterocolitica.

The sialidase inhibitors that can be used with the present inventioninclude, e.g., fetuin, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid(DANA) or a pharmaceutically acceptable salt thereof; ethyl(3R,4R,5S)-5-amino-4-acetamido-3-(pentan-3-yloxy)-cyclohex-1-ene-1-carboxylate);(2R,3R,4S)-4-guanidino-3-(prop-1-en-2-ylamino)-2-((1R,2R)-1,2,3-trihydroxypropyl)-3,4-dihydro-2H-pyran-6-carboxylicacid;(4S,5R,6R)-5-acetamido-4-carbamimidamido-6-[(1R,2R)-3-hydroxy-2-methoxypropyl]-5,6-dihydro-4H-pyran-2-carboxylicacid; and(1S,2S,3S,4R)-3-[(1S)-1-acetamido-2-ethyl-butyl]-4-(diaminomethylideneamino)-2-hydroxy-cyclopentane-1-carboxylicacid, or a pharmaceutically acceptable salt thereof.

One or more glycan-modifying agents can be added to the platelets. Suchglycan modifying agents include, for example, CMP-sialic acid, aCMP-sialic acid precursor, UDP galactose or a combination thereof. In anaspect, an enzyme that converts the CMP-sialic acid precursor toCMP-sialic acid can also be added to the platelets.

In certain embodiments, the platelets can be treated with at least oneglycan modifying agent in an amount effective to reduce the clearance ofthe population of platelets. In some embodiments, the glycan modifyingagent is selected from the group consisting UDP-galactose andUDP-galactose precursors. In some preferred embodiments, the glycanmodifying agent is UDP-galactose.

In certain embodiments, the platelets can be treated with certain sugarmolecules which lead to glycation of the exposed GlcNAc residues on GPIband thereby reduced platelet clearance, block platelet phagocytosis,increase platelet circulation time, and/or increase platelet storagetime.

In certain embodiments, the platelets can be treated with trehalose orother low molecular weight polysaccharides.

In certain embodiments, the platelets can be treated with proteaseinhibitors, such as matrix metalloprotease inhibitors.

In some embodiments, the in vivo circulation time of the platelets isincreased by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%,100%, 150%, 200%, or more

The platelets of the present invention can be stored chilled for atleast about 3 days, at least about 5 days, at least about 7 days, atleast about 10 days, at least about 14 days, at least about 21 days, orat least about 28 days.

Further, deriving platelets from stem cells in culture affords theopportunity to precondition the platelets and the megakaryocytes, andeven to genetically modify the stem cells or progenitor cells like themegakaryocytes in a manner that extends the shelf-life of the plateletsand enhances yield and viability after refrigeration and/orcryopreservation storage. For instance, the MKs can be engineered tohave altered levels of expression of genes involved in membrane lipidratios, protein glycosylation patterns, stress-induced proteins, 14-3-3ζ(translocation and the like.

In a further embodiment, the platelets are functional platelets. Inafurther embodiment the percentage of functional platelets is at leastabout 60%, is at least about 70%, is at least about 80% or is at leastabout 90%. In a yet further embodiment, the functional platelets areactive for at least 2 days when stored at 22-37° C.

Pharmaceutical Preparations

Exemplary compositions of the present disclosure may be formulationsuitable for use in treating a human patient, such as pyrogen-free oressentially pyrogen-free, and pathogen-free. When administered, thepharmaceutical preparations for use in this disclosure may be in apyrogen-free, pathogen-free, physiologically acceptable form.

Additional exemplary compositions of the present disclosure may beirradiated, e.g., prior to administration. For example, the cells may beirradiated with gamma irradiation, e.g., with a dosage of approximately25 gy. For example, the composition may be irradiated with a dosagesufficient to mitotically inactivate any nucleated eukaryotic cells,and/or pathogens contained in the composition, e.g., in a dosagesufficient to mitotically inactivate any pluripotent stem cells, MKs,leukocytes, and/or PVE-HE that may be contained therein.

Delivery of Platelets

Various membranes, devices and methods of their manufacture have beenproposed and evaluated as a means of transplanting cells and theirsecreted products into the human body and are collectively referred toin the patent literature as bioartificial implants. Typically they sharea common principle of operation, that is, the cells are sequesteredinside a chamber bounded by a semipermeable membrane. Long term cellviability is thought to rely on the sustained diffusive exchange ofnutrients and waste products with adjacent vascularized tissue. (U.S.Pat. Nos. 6,372,244, 6,113,938, 6,322,804 4,911,717, 5,855,613,6,083,523, 5,916,554, 6,511,473, 6,485,723). There are three major typesof devices described in the scientific and patent literature forimplantation of cells into various tissue compartments including: planardisk designs, hollow fiber-based designs, and geometric solid-baseddesigns. These devices are typically designed to be placed in a bodycavity.

Definitions

“Embryoid bodies” refers to clumps or clusters of pluripotent cells(e.g., iPSC or ESC) which may be formed by culturing pluripotent cellsunder non-attached conditions, e.g., on a low-adherent substrate or in a“hanging drop.” In these cultures, pluripotent cells can form clumps orclusters of cells denominated as embryoid bodies. See Itskovitz-Eldor etal., Mol Med. 2000 Feb.; 6(2):88-95, which is hereby incorporated byreference in its entirety. Typically, embryoid bodies initially form assolid clumps or clusters of pluripotent cells, and over time some of theembryoid bodies come to include fluid filled cavities, the latter formerbeing referred to in the literature as “simple” EBs and the latter as“cystic” embryoid bodies.

The term “embryonic stem cells” (ES cells) is used herein as it is usedin the art. This term includes cells derived from the inner cell mass ofhuman blastocysts or morulae, including those that have been seriallypassaged as cell lines. The ES cells may be derived from fertilizationof an egg cell with sperm, as well as using DNA, nuclear transfer,parthenogenesis, or by means to generate ES cells with homozygosity inthe HLA region. ES cells are also cells derived from a zygote,blastomeres, or blastocyst-staged mammalian embryo produced by thefusion of a sperm and egg cell, nuclear transfer, parthenogenesis,androgenesis, or the reprogramming of chromatin and subsequentincorporation of the reprogrammed chromatin into a plasma membrane toproduce a cell. Embryonic stem cells, regardless of their source or theparticular method used to produce them, can be identified based on (i)the ability to differentiate into cells of all three germ layers, (ii)expression of at least Oct 4 and alkaline phosphatase, and (iii) abilityto produce teratomas when transplanted into immunodeficient animals.Embryonic stem cells that may be used in embodiments of the presentinvention include, but are not limited to, human ES cells (“ESC” or “hEScells”) such as MA01, MA09, ACT-4, No. 3, H1, H7, H9, H14 and ACT30embryonic stem cells. Additional exemplary cell lines include NED1,NED2, NED3, NED4, NED5, and NED7. See also NIH Human Embryonic Stem CellRegistry. An exemplary human embryonic stem cell line that may be usedis MA09 cells. The isolation and preparation of MA09 cells waspreviously described in Klimanskaya, et al. (2006) “Human Embryonic StemCell lines Derived from Single Blastomeres.” Nature 444: 481-485. Thehuman ES cells used in accordance with exemplary embodiments of thepresent invention may be derived and maintained in accordance with GMPstandards.

As used herein, the term “pluripotent stem cells” includes embryonicstem cells, embryo-derived stem cells, and induced pluripotent stemcells, regardless of the method by which the pluripotent stem cells arederived. Pluripotent stem cells are defined functionally as stem cellsthat are: (a) capable of inducing teratomas when transplanted inimmunodeficient (SCID) mice; (b) capable of differentiating to celltypes of all three germ layers (e.g., can differentiate to ectodermal,mesodermal, and endodermal cell types); and (c) express one or moremarkers of embryonic stem cells (e.g., express Oct 4, alkalinephosphatase. SSEA-3 surface antigen, SSEA-4 surface antigen, nanog,TRA-1-60, TRA-1-81, SOX2, REX1, etc.). In certain embodiments,pluripotent stem cells express one or more markers selected from thegroup consisting of: OCT-4, alkaline phosphatase, SSEA-3, SSEA-4,TRA-1-60, and TRA-1-81. Exemplary pluripotent stem cells can begenerated using, for example, methods known in the art. Exemplarypluripotent stem cells include embryonic stem cells derived from the ICMof blastocyst stage embryos, as well as embryonic stem cells derivedfrom one or more blastomeres of a cleavage stage or morula stage embryo(optionally without destroying the remainder of the embryo). Suchembryonic stem cells can be generated from embryonic material producedby fertilization or by asexual means, including somatic cell nucleartransfer (SCNT), parthenogenesis, and androgenesis. Further exemplarypluripotent stem cells include induced pluripotent stem cells (iPSCs)generated by reprogramming a somatic cell by expressing a combination offactors (herein referred to as reprogramming factors). The iPSCs can begenerated using fetal, postnatal, newborn, juvenile, or adult somaticcells.

In certain embodiments, factors that can be used to reprogram somaticcells to pluripotent stem cells include, for example, a combination ofOct 4 (sometimes referred to as Oct 3/4), Sox2, c-Myc, and Klf4. Inother embodiments, factors that can be used to reprogram somatic cellsto pluripotent stem cells include, for example, a combination of Oct 4,Sox2, Nanog, and Lin28. In certain embodiments, at least tworeprogramming factors are expressed in a somatic cell to successfullyreprogram the somatic cell. In other embodiments, at least threereprogramming factors are expressed in a somatic cell to successfullyreprogram the somatic cell. In other embodiments, at least fourreprogramming factors are expressed in a somatic cell to successfullyreprogram the somatic cell. In other embodiments, additionalreprogramming factors are identified and used alone or in combinationwith one or more known reprogramming factors to reprogram a somatic cellto a pluripotent stem cell. Induced pluripotent stem cells are definedfunctionally and include cells that are reprogrammed using any of avariety of methods (integrative vectors, non-integrative vectors,chemical means, etc.). Pluripotent stem cells may be geneticallymodified or otherwise modified to increase longevity, potency, homing,to prevent or reduce alloimmune responses or to deliver a desired factorin cells that are differentiated from such pluripotent cells (forexample, platelets).

“Induced pluripotent stem cells” (iPS cells or iPSC) can be produced byprotein transduction of reprogramming factors in a somatic cell. Incertain embodiments, at least two reprogramming proteins are transducedinto a somatic cell to successfully reprogram the somatic cell. In otherembodiments, at least three reprogramming proteins are transduced into asomatic cell to successfully reprogram the somatic cell. In otherembodiments, at least four reprogramming proteins are transduced into asomatic cell to successfully reprogram the somatic cell.

The pluripotent stem cells can be from any species. Embryonic stem cellshave been successfully derived in, for example, mice, multiple speciesof non-human primates, and humans, and embryonic stem-like cells havebeen generated from numerous additional species. Thus, one of skill inthe art can generate embryonic stem cells and embryo-derived stem cellsfrom any species, including but not limited to, human, non-humanprimates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs(domestic and wild dogs), cats (domestic and wild cats such as lions,tigers, cheetahs), rabbits, hamsters, gerbils, squirrel, guinea pig,goats, elephants, panda (including giant panda), pigs, raccoon, horse,zebra, marine mammals (dolphin, whales, etc.) and the like. In certainembodiments, the species is an endangered species. In certainembodiments, the species is a currently extinct species.

Similarly, iPS cells can be from any species. These iPS cells have beensuccessfully generated using mouse and human cells. Furthermore, iPScells have been successfully generated using embryonic, fetal, newborn,and adult tissue. Accordingly, one can readily generate iPS cells usinga donor cell from any species. Thus, one can generate iPS cells from anyspecies, including but not limited to, human, non-human primates,rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic andwild dogs), cats (domestic and wild cats such as lions, tigers,cheetahs), rabbits, hamsters, goats, elephants, panda (including giantpanda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales,etc.) and the like. In certain embodiments, the species is an endangeredspecies. In certain embodiments, the species is a currently extinctspecies.

Induced pluripotent stem cells can be generated using, as a startingpoint, virtually any somatic cell of any developmental stage. Forexample, the cell can be from an embryo, fetus, neonate, juvenile, oradult donor. Exemplary somatic cells that can be used includefibroblasts, such as dermal fibroblasts obtained by a skin sample orbiopsy, synoviocytes from synovial tissue, foreskin cells, cheek cells,or lung fibroblasts. Although skin and cheek provide a readily availableand easily attainable source of appropriate cells, virtually any cellcan be used. In certain embodiments, the somatic cell is not afibroblast.

The induced pluripotent stem cell may be produced by expressing orinducing the expression of one or more reprogramming factors in asomatic cell. The somatic cell may be a fibroblast, such as a dermalfibroblast, synovial fibroblast, or lung fibroblast, or anon-fibroblastic somatic cell. The somatic cell may be reprogrammedthrough causing expression of (such as through viral transduction,integrating or non-integrating vectors, etc.) and/or contact with (e.g.,using protein transduction domains, electroporation, microinjection,cationic amphiphiles, fusion with lipid bilayers containing, detergentpermeabilization, etc.) at least 1, 2, 3, 4, 5 reprogramming factors.The reprogramming factors may be selected from Oct 3/4, Sox2, NANOG,Lin28, c Myc, and Klf4. Expression of the reprogramming factors may beinduced by contacting the somatic cells with at least one agent, such asa small organic molecule agents, that induce expression of reprogrammingfactors.

Further exemplary pluripotent stem cells include induced pluripotentstem cells generated by reprogramming a somatic cell by expressing orinducing expression of a combination of factors (“reprogrammingfactors”). iPS cells may be obtained from a cell bank. The making of iPScells may be an initial step in the production of differentiated cells.iPS cells may be specifically generated using material from a particularpatient or matched donor with the goal of generating tissue-matchedmegakaryocytes and platelets. iPSCs can be produced from cells that arenot substantially immunogenic in an intended recipient, e.g., producedfrom autologous cells or from cells histocompatible to an intendedrecipient.

The somatic cell may also be reprogrammed using a combinatorial approachwherein the reprogramming factor is expressed (e.g., using a viralvector, plasmid, and the like) and the expression of the reprogrammingfactor is induced (e.g., using a small organic molecule.) For example,reprogramming factors may be expressed in the somatic cell by infectionusing a viral vector, such as a retroviral vector or a lentiviralvector. Also, reprogramming factors may be expressed in the somatic cellusing a non-integrative vector, such as an episomal plasmid. See, e.g.,Yu et al., Science. 2009 May 8; 324(5928):797-801, which is herebyincorporated by reference in its entirety. When reprogramming factorsare expressed using non-integrative vectors, the factors may beexpressed in the cells using electroporation, transfection, ortransformation of the somatic cells with the vectors. For example, inmouse cells, expression of four factors (Oct3/4, Sox2, c myc, and Klf4)using integrative viral vectors is sufficient to reprogram a somaticcell. In human cells, expression of four factors (Oct3/4, Sox2, NANOG,and Lin28) using integrative viral vectors is sufficient to reprogram asomatic cell.

Once the reprogramming factors are expressed in the cells, the cells maybe cultured. Over time, cells with ES characteristics appear in theculture dish. The cells may be chosen and subcultured based on, forexample, ES morphology, or based on expression of a selectable ordetectable marker. The cells may be cultured to produce a culture ofcells that resemble ES cells—these are putative iPS cells.

To confirm the pluripotency of the iPS cells, the cells may be tested inone or more assays of pluripotency. For example, the cells may be testedfor expression of ES cell markers; the cells may be evaluated forability to produce teratomas when transplanted into SCID mice; the cellsmay be evaluated for ability to differentiate to produce cell types ofall three germ layers. Once a pluripotent iPSC is obtained it may beused to produce megakaryocyte cells and platelets.

The term “hemogenic endothelial cells” (PVE-HE) as used herein refers tocells capable of differentiating to give rise to hematopoietic celltypes or endothelial cell types, which may express PECAM1, VE-Cadherin,and/or endoglin (e.g., PECAM1+VE-Cad+Endoglin+hemogenic PVE-HE), andwhich may optionally be derived from pluripotent stem cells. These cellscan be described based on numerous structural and functional propertiesincluding, but not limited to, expression (RNA or protein) or lack ofexpression (RNA or protein) of one or more markers. The PVE-HE cells arecharacterized by the expression of the markers CD31 (PECAM 1). Forexample, at least about 90%, at least about 95%, or at least about 9addition, immunofluorescence and transmission electron microscopicresults further demonstrate 9% of the PVE-HE cells in a population maybe CD31+. The PVE-HE cells may also express the markers CD105(endoglin), and CD144 (VE-Cadherin). For example, at least about 70%,about at least about 80%, at least about 85%, at least about 90%, atleast about 95%, or at least about 99% of the PVE-HE cells in apopulation may be CD105+, CD144+, and CD31+. In certain embodiments thePVE-HE cells are loosely adherent to each other. CD31, the plateletendothelial cell adhesion molecule-1 (PECAM-1), has been used as amarker for the development of endothelial cell progenitors,vasculogenesis, and angiogenesis. CD31 is constitutively expressed onthe surface of adult and embryonic endothelial cells, is a majorconstituent of the endothelial cell intercellular junction (where up to10⁶ PECAM-1 molecules are concentrated) and is weakly expressed on manyperipheral leukocytes and platelets.

In exemplary embodiments, PVE-HE may exhibit one or more, preferablyall, of the following characteristics: (1) Significant amount ofCD31+PVE-HE cells can be detected as early as 72 hours after initiationof PVE-HE differentiation. (2) It will reach peak level at round 120 to146 hours after initiation of PVE-HE differentiation. (3) TheCD31+PVE-HE cell population can be isolated and cryopreserved. (3) Theyexpress almost all endothelial progenitor cell surface marker such asCD31, CD105 (Endoglin), CD144 (VE-Cadherin). They may also express CD34,CD309 (KDR) and CD146. (4) A subpopulation of CD31+PVE-HE cells alsoexpress CXCR4 (CD184). (5) The endothelial lineage potential can beconfirmed by culturing CD31+PVE-HE cells onto fibronectin in endothelialcell (EC)-specific medium (such as EGM-2 or EndoGro) to obtain monolayerof cells with typical endothelial morphology. (6) PVE-HE-derivedendothelial cells (PVE-HE-EC) not only express CD31 (localized atcell-cell junction), but also express von Willibrandt Factor (vWF), andcapable of LDL-uptake. (7) PVE-HE-ECs are capable of forming 3D-networkstructure when cultured on top of Matrigel. (8) When plating in extremelow density on Fibronectin in EC-specific medium, the CD31+PVE-HE cellsare capable of forming colonies with typical endothelial morphologyconfirming their clonogenic capability. (9) When plating inmethylcellulose medium for blast-colony growth, blast colony can only begenerated from CD31+ fraction. Unlike CD31− cells, both CD34- and CD105−are capable of generating blast colonies suggesting hemogenic capabilityis exclusively maintained in CD31 fraction only. (10) Newly derivedPVE-HE-EC maintain hemogenic potential and will give rise to hemogeniccells if cultured under conditions favorable to the hematopoieticlineages.

The term “megakaryocyte lineage-specific progenitor cells” (“MLPs”), asused herein, refers to mononuclear hematopoietic stem cells committed toat least the megakaryocyte lineage and includes, but is not limited to,cells in the umbilical cord blood, bone marrow, and peripheral blood aswell as hematopoietic stem cells, cells derived from human embryonicstem cells, and cells derived from induced pluripotent stem cells. Thesecells can be described based on numerous structural and functionalproperties including, but not limited to, expression (RNA or protein) orlack of expression (RNA or protein) of one or more markers. The MLPs ofthis disclosure can be a mixture of immature and mature cells. Thepercentage of mature vs. immature MLPs may vary based upon the length oftime in culture in MLP-Derivation and Expansion Medium (MLP-DEM, alsoreferred to as APEL) (described in Example 2). Immature MLPs arecharacterized by the expression of the markers CD41a, CD31, CD34, CD13and the lack of CD14 and CD42b marker expression. Exemplary methods ofthe disclosure provide for detection and/or purification of MLPs by amethod comprising detecting the expression of CD13 and/or enriching orpurifying CD13-positive cells. Optionally in these methods theexpression of CD13 may be detected and/or used as a basis for cellpurification in combination with the expression of one or moreadditional markers of immature or mature MLPs described herein. MatureMLPs are characterized by the expression of the markers CD41a, CD31,CD34, CD13, CD42b and the lack of CD14 expression. In certainembodiments MLPs generated in feeder free culture may be semi-detachedor detach completely and may float in the culture medium. For example,MLPs may be collected from the PVE-HE when they start to float up insuspension. Preferably MLPs are not plated and allowed to adhere, whichmay permit differentiation into lineages other than MKs or non-MKlineages (such as endothelial cells). MLPs are preferably grown insuspension to produce MKs. Optionally, MLPs may be cryopreserved.

The term “megakaryocytes” (MKs) as used herein refers to large polyploidhematopoietic cells that give rise to platelets, as well as smaller MKs(which may be produced by the subject methods) that may be diploid butfully capable of producing platelets. One primary morphologicalcharacteristic of mature MKs is the development of a large, polyploidnucleus. Mature MKs stop proliferating but continue to increase theirDNA content through endomitosis; with a parallel increase in cell size.The large polyploid nucleus, large cell volume, and ample cytoplasm ofMKs allows for the production of thousands of platelets per cell. MKscan be described based on these and numerous other structural andfunctional properties including, but not limited to, expression (RNA orprotein) or lack of expression (RNA or protein) of one or more markers.Mature MKs express the markers CD41a and CD42b. Mature MKs may alsoexpress CD61 and CD29. For example, a MK of the present disclosure ispreferably functional in the sense of being able to produce platelets,e.g., when cultured under conditions those described herein. Exemplaryembodiments provide a method of detecting matured MKs comprisingdetecting expression of CD29 and identifying CD29 positive cells asmatured MKs. Additional exemplary embodiments provide a method ofpurifying MKs, comprising purification of CD29 positive cells from apopulation, for example using magnetic bead subtraction, FACS, otherimmunoaffinity based methods, or the like. Optionally in these methodsthe expression of CD29 may be detected and/or used as a basis for cellpurification in combination with the expression of one or moreadditional markers of matured MKs, such as those markers shown in Table1 (which provides further exemplary cell surface marker expression ofmatured MKs).

In vivo, MK are derived from hematopoietic stem cell precursor cells inthe bone marrow. These multipotent stems cells reside in the marrowsinusoids and are capable of producing all types of blood cellsdepending on the signals they receive. The primary signal for MKproduction is TPO. TPO induces differentiation of progenitor cells inthe bone marrow towards a final MK phenotype. The MK develops throughthe following lineage: CFU-ME (pluripotent hemopoietic stem cell orhemocytoblast), megakaryoblast, promegakaryocyte, megakaryocyte. Thecell eventually reaches megakaryoblast stage and loses its ability todivide. However, it is still able to replicate its DNA and continuedevelopment, becoming polyploidy. The cytoplasm continues to expand andthe DNA complement can increase to greater than 64 N.

Once the cell has completed differentiation and becomes a maturemegakaryocyte, it begins the process of producing platelets. TPO plays arole in inducing the MK to form small proplatelet processes. Plateletsare held within these internal membranes within the cytoplasm of the MK.There are two proposed mechanisms for platelet release. In one scenario,these proplatelet processes break up explosively to become platelets.Alternatively, the cell may form platelet ribbons into blood vessels.The ribbons are formed via pseudopodia and they are able to continuouslyemit platelets into circulation. In either scenario, each of theseproplatelet processes can give rise to 2000-5000 new platelets uponbreakup. Overall, more than 75% of these newly-produced platelets willremain in circulation while the remainder will be sequestered by thespleen.

The term “platelet” as used herein refers to anucleate cytoplasmicbodies derived from cells that are involved in the cellular mechanismsof primary hemostasis leading to the formation of blood clots. Platelets(thrombocytes) are small, irregularly shaped clear cell fragments 2-3 μmin diameter, which in vivo are derived from fragmentation of precursormegakaryocytes (MK). Platelets can be identified based on numerousstructural and functional properties including, but not limited to,expression (RNA or protein) or lack of expression (RNA or protein) ofone or more markers. Platelets express the markers CD41a and CD42b.Platelets adhere to tissue and to each other in response to vascularinjury.

The terms “functional platelet” or “platelets that are functional” asused herein refer to platelets that can be activated by thrombin andparticipate in clot retraction. In exemplary embodiments, whetherplatelets are functional platelets may be determined using an animalmodel, e.g., as described in Example 4 (FIG. 12), for example bycomparison to naturally-derived platelets, which may be of the samespecies. Additionally, activated platelets can be identified byexpression of CD62p and αIIbβIII, and functional platelets can beidentified (optionally quantitatively) by their expression of thesemarkers upon activation such as upon activation with thrombin. Thephrase “substantially all of the platelets are functional” refers to acomposition or preparation comprising platelets wherein, for example, atleast 60% of the platelets are functional platelets, or at least 65%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% of the platelets are functionalplatelets. Functional platelets may be active for at least 5 days whenstored at 22-37° C.

PECAM1 (CD31) is a member of the immunoglobulin (Ig) superfamily that isexpressed on the surface of circulating platelets, monocytes,neutrophils, and particular T-cell subsets. It is also a majorconstituent of the endothelial cell intercellular junction, where up toan estimated 1 million molecules are concentrated. Because of thiscellular expression pattern, PECAM1 is implicated in several functions,including transendothelial migration of leukocytes, angiogenesis, andintegrin activation. Ig superfamily mediate cell adhesion (e.g., NCAM1,ICAM1, and VCAM1) or antigen recognition (e.g., immunoglobulins, T-cellreceptors, and MHC molecules). In addition, a subgroup comprising 30members characterized by the presence of 1 or more immunoreceptortyrosine-based inhibitory motifs (ITIMs) within their cytoplasmicdomains has also been recognized. PECAM1, which has 6 ITIMs within itscytoplasmic domain, is a member of this subfamily.

Endoglin (ENG), also called CD105, is a homodimeric membraneglycoprotein primarily associated with human vascular endothelium. It isalso found on bone marrow proerythroblasts, activated monocytes,fibroblasts, smooth muscle cells and lymphoblasts in childhood leukemia.Endoglin is a component of the transforming growth factor-beta (TGFB)receptor complex and binds TGFB1 with high affinity. Endoglin isinvolved in the cytoskeletal organization affecting cell morphology andmigration, in processes such as development of cardiovascular system andin vascular remodeling. Its expression is regulated during heartdevelopment. Experimental mice without the endoglin gene die due tocardiovascular abnormalities.

VE-cadherin (CD144) is a classical cadherin from the cadherinsuperfamily. VE-cadherin plays an important role in endothelial cellbiology through control of the cohesion and organization of theintercellular junctions, therefore maintain the integrity of theendothelium. VE-cadherin is indispensable for proper vasculardevelopment. Transgenic mouse models studies confirmed that VE-cadherindeficiency is embryonically lethal due to vascular defects. VE-cadherinserves the purpose of maintaining newly formed vessels.

The term “ROCK inhibitor” as used herein refers to any substance thatinhibits or reduces the function of Rho-associated kinase or itssignaling pathway in a cell, such as a small molecule, an siRNA, amiRNA, an antisense RNA, or the like. “ROCK signaling pathway,” as usedherein, may include any signal processors involved in the ROCK-relatedsignaling pathway, such as the Rho-ROCK-Myosin II signaling pathway, itsupstream signaling pathway, or its downstream signaling pathway in acell. An exemplary ROCK inhibitor that may be used is Stemgent'sStemolecule Y27632, a rho-associated protein kinase (ROCK) inhibitor(see Watanabe et al., Nat Biotechnol. 2007 Jun.; 25(6):681-6) Other ROCKinhibitors include, e.g., H-1152, Y-30141, Wf-536, HA-1077,hydroxyl-HA-1077, GSK269962A and SB-772077-B. Doe et al., J. Pharmacol.Exp. Ther., 32:89-98, 2007; Ishizaki, et al., Mol. Pharmacol.,57:976-983, 2000; Nakajima et al., Cancer Chemother. Pharmacol.,52:319-324, 2003; and Sasaki et al., Pharmacol. Ther., 93:225-232, 2002,each of which is incorporated herein by reference as if set forth in itsentirety. ROCK inhibitors may be utilized with concentrations and/orculture conditions as known in the art, for example as described in USPGPub No. 2012/0276063 which is hereby incorporated by reference in itsentirety. For example, the ROCK inhibitor may have a concentration ofabout 0.05 to about 50 microM, for example, at least or about 0.05, 0.1,0.2, 0.5, 0.8, 1, 1.5, 2, 2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45,or 50 microM, including any range derivable therein, or anyconcentration effective for promoting cell growth or survival.

For example, pluripotent stem cell viability may be improved byinclusion of a ROCK inhibitor. In an exemplary embodiment, thepluripotent stem cells may be maintained under feeder-free conditions.In another example, megakaryocyte lineage-specific progenitor cellviability may be improved by inclusion of a ROCK inhibitor. In anotherexample, megakaryocyte viability may be improved by inclusion of a ROCKinhibitor. In another exemplary embodiment, the megakaryocytelineage-specific progenitor cell may be maintained under feeder freeconditions.

ABBREVIATIONS

iPS: induced pluripotent stem

iPSC: induced pluripotent stem cell

hiPSC: human induced pluripotent stem cell

hES: human embryonic stem

hESC: human embryonic stem cell

MK: megakaryocyte

MLP: megakaryocytic lineage-specific progenitor also calledmegakaryocyte progenitor (MKP)

PVE-HE: hemogenic endothelial cell, which are optionallyPECAM1+VE-Cadherin+Endoglin+ and which are optionally derived frompluripotent stem cells,

iPS-PVE-HE: iPS cell-derived hemogenic endothelial cell (such as aPECAM1+VE-Cadherin+Endoglin+ cell)

hES-PVE-HE: hES cell-derived hemogenic endothelial cell (such as aPECAM1+VE-Cadherin+Endoglin+ cell)

PVE-HE-MLP: megakaryocytic lineage-specific progenitor produced from ahemogenic endothelial cell

iPS-PVE-HE-MLP: PVE-HE-MLP produced from an iPS cell

hES-PVE-HE-MLP: PVE-HE-MLP produced from an hES cell

PVE-HE-MLP-MK: megakaryocyte produced from a megakaryocyticlineage-specific progenitor which was produced from a hemogenicendothelial cell

iPS-PVE-HE-MLP-MK: PVE-HE-MLP-MK produced from an iPS cell

hES-PVE-HE-MLP-MK: PVE-HE-MLP-MK produced from an hES cell

PLT: platelet

hiPSC-PLT: platelet or platelet-like particle produced from humaninduced pluripotent stem cells

hESC-PLT: platelet or platelet-like particle produced from humanembryonic stem cells

ADM: advanced differentiation morphology.

REFERENCES

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All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1—Generation of Pluripotent-Derived Hemogenic Endothelial Cells(PVE-HE)

Pluripotent-Derived Hemogenic Endothelial Cells (PVE-HE) were generatedfrom induced pluripotent stem (iPS) cells.

First, iPS cells were expanded by culturing on Matrigel (a solublepreparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells) in thefeeder-free pluripotent stem cell culture medium mTeSR1. Briefly, humaniPS cells were harvested by dissociation using chemically defined CellDissociate Buffer (CDB) with EDTA as the sole active component. Noenzyme or any other animal products were used in the culture medium orCDB. For investigators with ordinary skill in the art, it should beunderstood that it is possible to use another chemically defined matrixsuch as recombinant vitronectin or SyntheMax II, or medium such asmTeSR2, or other compatible cell dissociation reagents.

Second, harvested human iPS cells were prepared for differentiation intomultipotent PVE-HE which were PECAM1+VE-Cadherin+Endoglin+. Embryoidbody (EB) formation was not required. Briefly, the harvested cells wereresuspended in mTeSR1, and plated on top of the extracellular matrixhuman collagen IV (Advanced BioMatrix, cat #5022). The small moleculeROCK inhibitor Y27632 was added to the culture at 10 μM and was thoughtto help the harvested iPS cells attach to the Collagen IV coatedsurface. The iPS cells were allowed to attach for 12-48 hours in at 37°C. with 5% CO2. As shown in FIG. 2A, after 48 hours attached cells showtypical pluripotent stem cell morphology under feeder-free condition.

Third, prepared human iPS cells were differentiated into PVE-HE cells.Briefly, mTeSR1 medium with Y27632 was removed and a differentiationinitiation medium (DIM) was added. DIM is an animal-component freemedium (ACF) comprised of Iscove's Modified Dulbecco's Medium (IMDM) asbasal medium, human serum albumin, iron-saturated transferrin, insulin,b-mercaptoethanol, soluble low-density lipoprotein (LDL), cholesterol,bone morphogenetic protein 4 (BMP4) at 50 ng/ml, basic fibroblast growthfactor (bFGF) at 50 ng/ml, and vascular endothelial growth factor (VEGF)at 50 ng/ml. After being incubated for 48 hours at 37° C. with 5% CO₂,early morphology of desired PVE-HE differentiation was observed. Thealmost complete transition from pluripotent stem cell morphology intoscattered small cell clusters can be seen in FIG. 2B. After 96 to 146hours post PVE-HE differentiation initiation, advanced differentiationmorphology was observed showing small compact cell clusters growing ontop of the monolayer (FIG. 2C).

Fourth, after 120 hours post PVE-HE differentiation initiation, advanceddifferentiation morphology (ADM) cells were analyzed for morphologicchanges (FIG. 3B) and successful PVE-HE differentiation. Briefly, asmall sample of cells was subjected flow cytometric analysis oflineage-specific markers CD31 (PECAM), CD105 (Endoglin), CD144(VE-Cadherin). The ADM cells show the PVE-HE phenotype CD31⁺CD144⁺CD105⁺at this stage of differentiation (FIG. 3A).

Example 2—Generating Detached Megakaryocytic Lineage-SpecificProgenitors (MLPs) from Human iPS-Derived PVE-HE Cells

First, initiation of MLP differentiation was performed 120 hours afterinitiating PVE-HE differentiation. Briefly, DIM medium containing 50ng/ml BMP4, 50 ng/ml bFGF, and 50 ng/ml VEGF was removed and replacedwith MLP Derivation and Expansion medium (MLP-DEM, also referred to asAPEL or Stemline II, as shown in FIG. 1) comprised of Iscove's modifiedDulbecco's medium (IMDM), Ham's F-12 nutrient mixture, Albucult (rhAlbumin), Polyvinylalcohol (PVA), Linoleic acid, SyntheChol (syntheticcholesterol), Monothioglycerol (a-MTG), rhInsulin-transferrin-selenium-ethanolamine solution, protein-freehybridoma mixture I (PFHMII), ascorbic acid 2 phosphate, Glutamax I(L-alanyl-L-glutamine), Penicillin/streptomycin, Stem Cell Factor (SCF)at 25 ng/ml, Thrombopoietin (TPO) at 25 ng/ml, Fms-related tyrosinekinase 3 ligand (FL) at 25 ng/ml, Interleukin-3 (IL-3) at 10 ng/ml,Interleukin-6 (IL-6) at 10 ng/ml, and Heparin at 5 Units/mi. Cells werethen incubated for up to 8 days at 37° C. with 5% CO2. MLPdifferentiation was maintained for 8 days.

TABLE 1 MLP/MK marker comparison Matured MLPs Matured MKs CD43 95.9%98.5% CD41 89.3% 91.4% CD61 77.2% 89.7% CD42a 66.3% 88.8% CD144 57.6%15.1% CD31 57.0% 80.6% CD29 40.4% 96.2% CD45 40.3% 66.1% CD13 40.4%59.4% CD34 30.3% 44.1% CD309 28.4% 73.2% CD71 26.4% 59.6% CD90 19.8% 2.0% CD105  7.5% 46.0% CD56  3.5%  3.3% CD14  4.4%  4.7% CD143  2.2% 6.8% CD15  1.6% 11.0% CD3  2.8%  4.2% CD117  2.7% 10.2% CD184  0.6% 8.5% CD11c  0.4%  2.5% CD73  0.0%  1.5%

Table 1 shows comparative flow cytometric analysis of marker expressionby matured MLP and matured MK cells. The analysis characterizes thephenotype of PVE-HE-derived MLP cells prior to cryopreservation andsubsequent differentiation into MK cells of the defined phenotype.

Second, floating and semi-detached MLPs on top of the attached cellpopulation were collected by wash with mild force using serologicalpipette. Medium containing these MLPs was transferred into conical tubesand centrifuge at 300×g for 5 minutes to collect the MLPs. Medium wasdiscarded and MLP pellet was resuspended in phosphate buffered salineand analyzed for morphology (FIG. 5) and by flow cytometry using markersseen in Table 1. Selected results seen in FIG. 4. Results demonstratethat MLPs collected at this stage are mainly (over 90%) comprised of twopopulations, one characterized by a relatively immature MLP populationrepresented by CD41a⁺CD31⁺CD34⁺CD14⁻CD13⁺CD42b⁺, and a more matured MLPspopulation represented by CD41a⁺CD31⁺CD34⁺CD14⁻CD13⁺CD42b⁺.

Third, MLP cells were cryopreserved. Briefly, cryopreservation of MLPswas achieved using the cell freezing medium CS10 (Sigma) containing 10%DMSO.

Example 3—Generating Mature Platelets from Human iPS-PVE-HE-MLP-DerivedMegakaryocytes (MK)

First, initiation of platelet differentiation was performed using humaniPS-PVE-HE-derived MLP cells, as described above. MLPs were seeded ontoa non-adherent surface in MK media (MK-M) comprised of Iscove's ModifiedDulbecco's Medium (IMDM) as basal medium, human serum albumin,iron-saturated transferrin, insulin, b-mercaptoethanol, solublelow-density lipoprotein (LDL), cholesterol, TPO at 30 ng/ml, SCF at 1ng/ml, IL-6 at 7.5 ng/ml, IL-9 at 13.5 ng/ml, Y27632 at 5 μM, andHeparin at 5-25 units/ml. Cells were then incubated at 37° C. in 5% CO2for 3 days.

In some instances, the MLPs were seeded onto a non-adherent surface inculture medium comprised of Iscove's modified Dulbecco's medium (IMDM),Ham's F-12 nutrient mixture, Albucult (rh Albumin), Polyvinylalcohol(PVA), Linoleic acid, Linolenic acid, SyntheChol (syntheticcholesterol), Monothioglycerol (a-MTG), rhInsulin-transferrin-selenium-ethanolamine solution, protein-freehybridoma mixture II (PFHMII), ascorbic acid 2 phosphate, Glutamax I(L-alanyl-L-glutamine), Penicillin/streptomycin, TPO at 30 ng/ml, SCF at1 ng/ml, IL-6 at 7.5 ng/ml, IL-9 at 13.5 ng/ml, Y27632 at 5 μM, andHeparin at 5-25 units/ml. Cells were then incubated at 37° C. in 5% CO2for 3 days.

Second, iPS-PVE-HE-MLP-derived maturing MK cells were analyzed. At 72hours post initiation of platelet differentiation, very large polyploidMKs (50 μM) became abundant with progression of maturation (FIGS. 5A &B. Scale bar in 5A is 100 μM, N in 5B indicates nucleus inside MK). Frombetween 72-96 hours proplatelet forming cells with elongated pseudopodiawere readily observed microscopically. (FIGS. 5A & 5C, indicated byarrows), and emerging amounts of CD41a+CD42b+ platelet particles weredetected using flow cytometric analysis (FIG. 6). In FIG. 6 the cells in“A” were circulating human platelets, “B” is hES-PVE-HE-MLP, “C” isperipheral blood derived human platelets, “D” is iPS-PVE-HE-MLP, and “E”is hES-PVE-HE-MLP). At 84 hours post initiation, the amount ofCD41a+CD42b+ platelets increased dramatically, reaching levels as highas 70% (FIG. 6D).

Third, high quality platelets were harvested without harming maturingMKs in cultures for at least 3-5 consecutive days and possibly longer,maximizing the yield of platelets from MLPs for at least 3-5 times. Toseparate large MKs from platelet containing media, the cell suspensionwas centrifuged at 50×g for 10 minutes, supernatant removed and MK cellpellets re-suspended with fresh MK-M, allowing them to produce moreplatelets later. To separate platelets from proplatelets, large celldebris and small MKs, a BSA/HSA gradient sedimentation method wasapplied to obtain a purer population of platelets from the supernatantof the 50×g centrifugation. Purified platelets were suspended with MK-Mand maintained at room temperature. Quality of platelets with respect togranularity, transparency, size, and cell surface marker expression werecharacterized by FACS analysis and compared to peripheral blood derivedhuman platelets. (FIG. 6A-E). Purified platelets were stored in MK-Mmedia at room temperature within a non-adherent surface, securing aminimal loss of functional platelets.

Example 4—Analysis of Mature Platelets from iPS-PVE-HE-MLP-DerivedMegakaryocytes (MK)

First, iPS-PVE-HE-MLP-derived platelets (hiPSC-PLTs) were analyzed formorphology. Immunoflouresensce and transmission electron microscopicanalysis was performed according to methods previously described (CellResearch 201121:530-45). hiPSC-PLTs were found to be discoid and mostlyultrastructurally identical to circulating human PLTs (as demonstratedby transmission electron microscopy, FIG. 7). hiPSC-PLTs were comparableon average size with circulating human PLTs (2.38 μm+0.85 μm versus 2.27μm+0.49 μm) as demonstrated by DIC and β1-tubulin IF microscopy (FIG.8). hiPSC-PLTs spread on glass—form both filopodia and lamelopodia (asdemonstrated by DIC live-cell microscopy images shown in FIG. 9).hiPSC-PLTs were anucleate, and comparable to circulating human PLTs (asdemonstrated by Hoechst staining, FIG. 8). hiPSC-PLTs were seen to havenormal tubulin cytoskeleton relative to circulating human PLTs (asdemonstrated by β1-tubulin labeling, FIG. 8). hiPSC-PLTs were seen tohave normal filamentous actin relative to circulating human PLTs (asdemonstrated by phalloidin labeling, FIG. 8). hiPSC-PLTs were seen tohave normal alpha-granule expression relative to circulating human PLTs(as demonstrated by TSP4 and PF4 labeling in FIGS. 10A & 10B).

Second, hiPSC-PLTs were analyzed for functionality using an in vitroactivation assay measuring the cell adhesion molecule expression on anactivated platelet. Briefly, two adhesion molecules were analyzed, usingan anti-CD62p antibody, and the PAC-1 antibody. PAC-1 recognizes theαIIbβIII integrin. Both CD62p and αIIbβIII are expressed on the surfaceof activated platelets. The assay was performed according to methodsdescribed previously (Cell Research 201121:530-45). In response tothrombin exposure, both PAC-1 and P-selectin binding were increased(FIG. 11).

Third, hiPSC-PLTs were analyzed for functionality using an in vivo assaysystem measuring thrombus formation in macrophage-depleted NOD/SCIDmice. Briefly, intravital microscopy of cremaster muscle arterioles wasperformed as previously described (Cell Research 201121:530-45).Macrophage-depleted NOD/SCID mice were anesthetized by intraperitonealinjection of ketamine (125 mg/kg) and xylazine (25 mg/kg). A trachealtube was inserted and the mouse was placed on a thermo-controlledblanket. After incision of the scrotum, the cremaster muscle wasexteriorized onto an intravital microscopy tray. The muscle preparationwas superfused with thermo-controlled (37° C.) and aerated (95% N2, 5%CO2) bicarbonate-buffered saline throughout the experiment. Thecremaster muscle arteriolar wall was injured by micropoint laserablation using a Micropoint Laser System (Photonics Instruments). Thedeveloping mouse platelet thrombus was visualized by infusion of Dylight649-conjugated anti-mouse CD42c antibodies (Emfret Analytics, 0.05 μg/gbody weight) through a jugular cannulus. Calcein AM-labeled hPLT,iPSC-PLT, and ESC-PLT, 3×10⁶, were also infused with or without ReoPro,100×g, into mice. Two to four thrombi were generated in one mouse.Fluorescence and brightfield images were recorded using an Olympus BX61Wmicroscope with a 60×/1.0 NA water immersion objective and a high speedcamera (Hamamatsu C9300) through an intensifier (Video ScopeInternational). Data were collected for 5 min following vessel wallinjury and analyzed using Slidebook v5.0 (Intelligent ImagingInnovations). The results seen in FIG. 12A are an indication thathiPSC-PLTs contribute to clot formation in an in vivo setting.hiPSC-PLT's functional capabilities were also determined to be mediatedby αIIbβIII. Specifically, hiPSC-PLTs were pretreated with ReoPro, a Fabfragment of a human-murine chimeric monoclonal antibody that bindsspecifically to αIIbβIII and inhibits platelet function (FIG. 12B).Asterisks indicate a statistically significant difference compared tocontrols not treated with ReoPro (p<0.01 versus control, Student'st-test).

Fourth, the kinetics of hiPSC-PLTs in macrophage-depleted NOD/SCID miceafter infusion were determined. To determine whether the kinetics ofhiPSC-PLT and ESC-PLT is similar to that of hPLT in vivo, iPSC- orESC-PLTs were infused into macrophage-depleted NOD/SCID mice, and bloodcollected at various time points was analyzed by flow cytometry.Briefly, macrophages were depleted by intravenous injection ofliposome-encapsulated clodronate as described previously.Clodronate-liposomes were injected into mice through a tail vein at Day0 (100 μl) and Day 2 (50 μl). At Day 3, human platelet-rich plasma wasobtained by centrifugation of sodium citrate-treated blood at 200×g for25 min and centrifuged at 800×g for 10 min in the presence of 0.5 μMPGE1 and 10% citrate buffer. The pellet was resuspended withHEPES-Tyrode buffer containing 0.15 μM PGE1 and 10% citrate buffer andcentrifuged at 800×g for 5 min. Platelets were resuspended inHEPES-Tyrode buffer containing 0.1% fatty acid-free BSA. Isolated humanplatelets (hPLT, 1.5×10⁷), induced pluripotent stem cell-derivedplatelets (hiPSC-PLT, 1.5×10⁷) of the present disclosure, and embryonicstem cell-derived platelets (ESC-PLT, 1.0×10⁷) of the present disclosurewere intravenously infused into macrophage-depleted mice. Mouse blood,30 μl, was collected through a jugular vein at different time points(10, 30, 60, 120, 240, 360, and 480 minutes) and analyzed by flowcytometry using APC-conjugated anti-human CD41 and Dylight488-conjugated anti-mouse CD42c antibodies. Results demonstrate thathiPSC-PLTs circulate for several hours in macrophage-depleted NOD/SCIDmice (FIG. 13).

Example 5. Production of Platelets from Pluripotent Cells

This example provides further exemplary methods of producing plateletsfrom pluripotent stem cells.

As noted above, hESC-PLT and hiPSC-PLT are human platelets which areproduced ex vivo from human embryonic and induced pluripotent stemcells. Initial in vitro and in vivo characterization described above hasdemonstrated that both hESC-PLT and hiPSC-PLT are morphologically andfunctionally comparable to human donor platelets. For example, hiPSC-PLTwere able to spread on a glass substrate.

The production of bulk intermediate (MK progenitors) is initiated withthe thaw of vials from an approved hiPSC or hESC master cell bank (MCB).The process flow chart for the production of bulk intermediate ispresented in FIG. 14A-E. Cryovials containing 1-2 million hiPSC or hESCmaster or working cell banks are removed from the vapor phase of cGMPliquid nitrogen storage and transferred to the clean room (ISO Class 7).All processing is performed in a certified biosafety cabinet (ISO Class5 BSC). Following thaw and wash to remove DMSO; cells are seeded onMatrigel coated vessels in mTeSR1 defined culture media. Care is takento maintain the cells as aggregates. Plates are labeled with the date,lot number, and passage number and the lid of each well is labeled aunique number (e.g. 1-6). Seeded plates are placed in a 37° C., 5% COincubator. Cultures are inspected daily using a stereo-microscope and aninverted light microscope. Observations regarding colony size, cellularmorphology; including the extent of differentiation, and media color arerecorded on a work sheet. mTeSR1 medium is changed typically every 1-2days until cultures are 60-90% confluent. When morphology and confluenceevaluations indicate that the cultures need to be passaged, colonies areharvested using cell dissociation buffer. Care is again taken tomaintain the cells as aggregates. Cultures are re-seeded onto Matrigelcoated vessels in mTeSR1 media. Based on the cm² harvested and the cm²to be seeded, cells are typically split at a ratio of 1:4 to 1:8 every3-7 days depending on the yield of stem cells required. Seeded culturesare returned to a 37° C., 5% CO₂ incubator. Stem cells may be passed 1-6times before induction of hemogenic differentiation depending on therequired lot size.

Three day post-seeding for hemogenic cell differentiation, cultures areexamined to confirm cell attachment and outgrowth. At this stagecultures are typically low density with attached cells covering lessthan 10% of the total surface area. Well attached cells shoulddemonstrate outgrowth with a significant transition from pluripotentstem cell morphology to differentiated cells: larger cells withsignificantly more cytoplasm relative to the size of the nucleus growingmore diffusely with no clear colony borders.

When the stem cell expansion phase is estimated to meet cell yieldrequirements, hemogenic differentiation is initiated. A representativevessel is sacrificed for harvest and a single cell suspension iscreated. The cell concentration is quantified to establish cultureseeding parameters, with the remaining cells discarded. Colonies arecarefully harvested using cell dissociation buffer and reseeded intocollagen IV coated vessels at a density of 5,000 cells/cm in mTeSR1media supplemented with 10 uM Y27632 (ROCK inhibitor). Seeded vesselsare placed in a 37° C., 5% CO₂ incubator.

The following day the media is removed, taking care to minimize theremoval any floating cell clusters, and replaced with StemSpan ACF(StemCell Technologies Inc.) supplemented with recombinant human (rh)BMP-4 at 50 ng/ml, rh VEGF at 50 ng/ml, and rh bFGF at 50 ng/ml. Thecultures are transferred to a low O₂ (˜5%), 37° C., 5% CO₂ environmentand are left undisturbed for 2 days. After 2 days the cultures areevaluated morphologically for cell attachment and outgrowth. The mediais removed, taking care to minimize the removal any floating cellclusters, and replaced with fresh media. The cultures are returned tothe low O₂ (˜5%), 37° C., 5% CO₂ environment for an additional 2 days.Following this time period the media is changed once more and thecultures are placed in normoxic culture conditions. After 2 days ofnormoxic culture the culture media is removed, taking care to minimizethe removal of any floating cell clusters, and replaced with MLP mediumcomprising STEMdiffAPEL (StemCell Technologies Inc.) supplemented with 5Units/ml heparin, rh 25 ng/ml TPO, 25 ng/ml rh SCF, 25/ng rh FL, rh IL-6and rh IL-3. These conditions are maintained for the next 2-6 days.Cultures undergo daily morphological evaluation to assess the quality offloating MK progenitors. Media changes are not performed, but additionalmedia is added if the culture begins to indicate media depletion(culture media appears yellow). Cultures are periodically sampled andassayed by FACS for CD41a, CD42b expression. When culture morphology andCD41a, CD42b expression (˜≥15% double positive) are appropriate, thecultures are harvested Cells are then cryopreserved @ 3-5 million viableMLPs/mL/cryovial in 10% dimethyl sulfoxide, 900 fetal bovine serumcryopreservation medium (Hyclone). Cryopreservation is accomplished byplacing vials in freezing container (Nalgene) and then storing in a −80°C. freezer for 1-3 days, followed by transfer to the vapor phase of thecGMP liquid nitrogen storage system.

The production process for the final product hESC-PLT or hiPSC-PLT isinitiated with the thaw of approved bulk intermediate. Cryovialscontaining 3-5 million MLPs which have passed bulk intermediate qualitytesting are removed from the vapor phase of cGMP liquid nitrogen storageand transferred to the clean room. Cells are seeded into ultra-lowattachment (ULA) vessels at approximately 2.3×10⁵/cm² in StemSpan ACFmedia supplemented with 30 ng/ml rh TPO, 1 ng/ml rh SCF, 7.5 ng/ml rhIL-6, 13.5 ng/ml rh IL-9 and 5 uM Y27632 (ROCK inhibitor). Culturevessels are labeled with the date and lot number and the lid on eachvessel is labeled a unique number (e.g. 1-6). Cultures are maintained at39° C., in a humidified 10/CO₂ atmosphere. Typically there is nosignificant cell expansion during this culture phase. Approximately 3 to4 days into the culture, large maturing megakaryocytes (MK) will becomeevident. Cultures are monitored by periodic sampling and FACS analysisfor CD41a, CD42b expression. Proplatelet formation is typically firstobserved after five days in culture. As proplatelet formation progressesand CD41a, CD42b expression increases to approximately 30%-70% thecultures can be harvested (days 6-7).

The harvested media is centrifuged at 50×g to remove the megakaryocyteslurry. The supernatant is removed and centrifuged at 1000×g toconcentrate the platelets. The platelets are resuspended and then loadedon to a discontinuous albumin (Human) (HSA) gradient (12%, 10%, 7%, 5%and 2%) to further isolate the platelets. The platelet HSA gradient iscentrifuged at 80×g for 15 minutes. Platelets are harvested from thegradient and PGE₁ is added to the suspension to prevent activation.Platelets are concentrated by centrifugation at 1000 g for 10 minutes.Currently hESC and hiPSC platelets are stored in culture media. For thepurposes of the proposed study, a target product stability of 48 to 72hours will be investigated. Preliminary studies indicate a minimum of 24hour stability as determined by PAC1 binding results pre and poststorage.

FACS analyses (CD31 and CD43) of the MLP cell population in the bulkintermediate have shown that approximately 98% of the cells arecommitted to a hemogenic endothelial or hematopoietic lineage and thushave differentiated beyond pluripotency.

Further downstream in the process, cells present during MKdifferentiation and PLT harvest phase of manufacture have been tested byvWF (von Willebrand Factor) expression. Cells at this phase ofmanufacture are approximately 100% vWF+. The maintenance of a highlydifferentiated cell population indicates that the culture is notexperiencing a clonal expansion of undifferentiated progenitors.

Preliminary studies indicate a total absence of pluripotent cells inhiPS-derived MK cell populations analyzed by IFA staining for OCT4,NANOG, TRA-1-60, TRA-1-81 SSE3, SSE4 and Alkaline Phosphatase.Additional studies using IFA staining for pluripotent markers willexamine populations of hES-derived and hiPS-derived MLPs and MKs, aswell as, purified PLTs derived from the MKs, to screen for the presenceof pluripotent cells. Spiking studies will be conducted to determine theLODs of this assay to detect stem cells in the various cell populations.

Preliminary studies have been performed to characterize MK populationsusing FACs analyses for pluripotent markers (SSEA4 and TRA-1-60).Spiking studies have been performed where hES cells were mixed with MKsat 1% and 0.1% concentrations. The results indicate a limit of detectionof 0.1%. An initial study characterizing MKs for SSEA4 and TRA-1-60provided a result of <0.1% (below the assay level of detection)SSEA4/TRA-1-60 positive cells in the MK population.

Referring to FIG. 14, steps 2-3, since stem cells harvested withdissociation buffer are dislodged as cell clumps, accurate cell countsare not possible. For this reason, a representative culture vessel isharvested using Trypsin-EDTA to ensure a single cell suspension. Thissuspension is counted in a hemocytometer and the harvested cell numberis normalized per cm². Cells used for counting are then discarded. Theremaining product cultures are harvested using dissociation buffer andthe cell yield is calculated based on the normalized cells/cm²×cm²harvested. The calculated cell yield is used to set the required celldensity/cm² for seeding vessels to induce hemogenic celldifferentiation.

Referring to FIG. 14, step 10, beginning at 2 days in MLP medium and outto 6 days in MLP medium cell samples are taken from representativeculture vessels, pooled and assessed for the percentage of CD41a andCD42b doubly positive cells by FACS analysis. CD41a is a subunit offibrinogen receptor (αIIbβIII) and CD42b is a subunit on von WillebrandFactor receptor (GPIb-V-IX). The expression of both receptors isspecific for MK lineages and both are required for platelet function.Early lineage hemogenic endothelial cells are CD41a negative, expressingCD41a during late-stage hemogenic differentiation in hematopoieticprogenitors. CD42b is expressed exclusively in mature MKs. At thispoint, cultures are heterogeneous with a high percentage of CD41+ cellsand a low percentage of CD42+ cells (Pineault, et. Al., Megakaryocyteand platelet production from human cord blood stem cells. Methods MolBiol. 2012; 788:219-47).

The sample preparation and FACS analysis are performed as follows.Briefly, floating cells are collected and pooled. Using a serologicalpipette a gentle stream of growth medium is directed towards the culturesurface to detach any loosely adherent MLPs and pooled with thefree-floating cells. A sample (100-200 μL) of well-suspended pooledcells is collected and centrifuged (160×g for 5 minutes). The pellets isresuspended in DPBS, centrifuged again, and resuspended in DPBS plus 3%FBS (FACS buffer) containing CD41a-APC-conjugated (allophycocyanin) andCD42b-PE-conjugated (phycoerythrin) (BD Bioscience, San Jose, Calif.).Fluorescent conjugated antibodies and the appropriate isotype controls(mouse IgG1k-APC and mouse IgG1k-PE) are incubated in the presence for15 minutes at room temperature. Labeled cells are then diluted in FACSbuffer, centrifuged and resuspended in FACS buffer. FACS analysis isperformed by monitoring 10,000 events. Cultures with an acceptablepercentage of cells expressing double positive (CD41a+CD42b+) MLPs areharvested and cryopreserved. The tentative minimum specification is 10%double positive cells. Shown in FIG. 15 is a representative twodimensional dot plot for MLPs derived from hiPSC. In that Figure thecell population is 86.4% CD41a+; 31.2% CD42b+ with 29.9% of thepopulation staining double positive.

Prior to harvesting for cryopreservation, MLP cultures are assessed forthe approximate percentage of attached cells and the extent ofdifferentiated large cells with low nuclei to cytoplasm ratio. Attachedcells should appear as diffuse colonies with no clear colony borders.There should be an abundance of the floating MLPs resting on top of theattached cell population. Viable floating MLPs should appear clear, withminimal birefringence, demarked by a smooth cell membrane. Typically, asurface area of 1 cm² generates 5,000 to 15,000 MLPs per day. Shown inFIG. 16 is microphotograph of a representative population of MLPsderived from hiPSC line MA-iPS-01 (Hoffman Modulation Contrast, ×400)

Prior to cryopreservation MLPs, viable cell count is performed by trypanblue exclusion using a hemocytometer. Cells are assessed for the viablecell number and the percent viability. Representative vials are testedfor mycoplasma and sterility.

Additionally, a representative vial of cryopreserved MLPs is assessedfor CD31 and CD43 expression, as follows. CD31 is a marker for hemogenicendothelial cells expressed in both endothelial and hematopoieticlineages. Expression of CD43 confirms hematopoietic commitment. A samplevial of cryopreserved MLPs is thawed rinsed, resuspended in DPBS plus 3%FBS (FACS buffer) and centrifuged (160×g for 5 minutes). The pellet isresuspended in in DPBS plus 3% FBS (FACS buffer) containingCD31-APC-conjugated (allophycocyanin) and CD43-FITC-conjugated (BDBioscience, San Jose, Calif.). Samples of thawed MLPs are incubated withthe fluorescent conjugated antibodies and the appropriate isotypecontrols (mouse IgG-APC and mouse IgG-FITC) for 15 minutes at roomtemperature. Labeled cells are then diluted in FACS buffer, centrifugedand resuspended in FACS buffer. FACS analysis is performed by monitoring10,000 events. Acceptable MLP banks have >/=50% CD31 positive cellsand >/=50% CD43 positive cells. Shown in FIG. 17 is a representative twodimensional dot plot for MLPs derived from hiPSC line MA-iPS-01. In theexample shown, the cell population is 99.8% CD31+; 98.5% CD43+ with98.4% of the population staining double positive.

Additionally, a representative vial of cryopreserved MLPs is assessed byFACS analysis utilizing pluripotent markers such as SSEA4, TRA-1-60 toconfirm the absence of pluripotent cells.

Sample cryovials of cryopreserved MLPs are thawed and assessed forviability and recovery post-thaw. MLPs are further processed to thepoint of proplatelet formation and assessed for morphology MK morphologyand FACS analysis for CD41a+ and CD42b+ during MK maturation. Culturesare monitored for proplatelet formation and for platelet formation andcharacterization by FACS analysis for doubly stained (CD41a+ and CD42b+)cells. Acceptance criteria include: sterility (negative on <USP 21>Immersion test), negative for mycoplasma (tested by Direct (agar &broth), Indirect (cell culture)), at least 90% positive for CD31 andCD43 by FACS, at least 70% viability by Trypan Blue, and negative forexpression of pluripotent cell markers.

Referring to FIG. 14, steps 13-14, after 2-3 days post-thaw and seedingof MLPs cultures are assessed for emergence of free-floating cells inthe MK lineage. At this time cells are heterogeneous in size rangingfrom 10-50 microns in diameter. The approximate proportion of viablecells is assessed by visual observation in an inverted light microscopewith healthy MK precursor cells and MKs displaying bright cytoplasm andsmooth cellular membranes. Shown in FIG. 18 is microphotograph of arepresentative population of MKs derived from hESC (Hoffman ModulationContrast, ×400)

Referring to FIG. 14, steps 13-14, after 2-3 days post-thawing of MLPs,cell samples are removed from representative culture vessels, pooled,processed, labeled with fluorescent-conjugated antibodies to CD41a andCD41b, and undergo FACS analysis. Cultures with an acceptable percentageof cells expressing both CD41a+ and CD42b+ MLPs are processed further(preferably at least 10% double positive cells).

Referring to FIG. 14, step 15, beginning at 3 days and out to 5 dayspost-thawing of MLPs, MK cultures are assessed for the appearance ofproplatelets. The arrows in FIG. 19 depict proplatelet morphology. MKdisplaying proplatelets exhibit long projections extending from thecells showing some beading and branching prior to fragmentation.

Referring to FIG. 14, steps 15-16, upon confirming the presence ofproplatelets, beginning at 3 days and out to 8 days post-thawing ofMLPs, samples of proplatelets and platelets are collected fromrepresentative cultures. Briefly, using a 10 mL serological pipette, thesamples are transferred to a 50 mL conical tube and drawn up andexpelled at least 5 times to generate shear force sufficient to fracturethe proplatelets. Samples are returned to a 39° C. incubator gassed with10/CO₂ and allowed to settle for 30 minutes. Approximately 250 μL of thesupernatant containing suspended platelets is stained and undergoes FACSanalysis for CD41a and CD42b. Platelet harvesting and subsequentprocessing is initiated when peak levels of platelets are detected,typically when 30-70% of the platelets are double positive stained(CD41a+CD42b+).

hiPSC-PLT and hESC-PLT are characterized for pH, platelet count andidentity (determined by expression of CD61, CD41a, CD42b), expression ofplatelet activation markers (CD62P, PAC1, Platelet Factor 4),Physiologic responses (Aggregation (microplate assay), TEG(thromboelastography), and Spreading), and morphologically evaluated byElectron Microcopy as well as DIC Microscopy with B1 Tubulin staining.

Cells are further tested for sterility (negative result of <USP 21>Immersion test), endotoxin (Gel Clot USP <85> less than 5 EU/ml),mycoplasma (negative result by European Pharmacopoeia and USPharmacopoeia test), identity (by FACS, at least 70% CD41a+, CD42b+),PLT Count/mpPLT (FACS for CD61), morphology (DIC Microscopy with β1Tubulin Staining), and potency by PAC1 Binding (Activated).

Purified platelets are stained with fluorescent-conjugated antibodies toCD41a and CD41b and undergo FACS analysis as described above. Shownbelow are representative two dimensional dot plot of PLTs derived fromiPS-01. In the example shown in FIG. 20, the population is 81.8% CD41a+;69.2% CD42b+ with 68.2% of the population staining double positive.

In conjunction with CD41a+,CD42b+ FACS performed in FIG. 20, propidiumiodine (P) is added to the sample during CD staining to assessviability. FACS analysis is performed counting 10,000 events asdescribed previously, with additional events collected at flow channel 3to detect PI positive events (nonviable platelets). Data are analyzed togive the percentage of CD41a+/CD41b+ platelets and the percent viability(Total Platelets Detected−PI+Platelets/Total Platelets Detected).

Each final product lot of platelets is assessed for the number ofplatelets and the number of platelet microparticles by detecting CD61positive events using flow cytometric analysis. This assay is performedusing a known number of fluorescent beads to normalize data and obtainthe absolute number of PLTs/μL and mpPLTs/μL. Briefly, 5-10 μL samplesof platelets are diluted in 0.9% saline and dispensed into each of twotubes: 1) one TruCount (BD Cat #340334) tube containing a lyophilizedpellet with a known number of fluorescent beads andphycoerythrin-conjugated anti-CD61 IgG in a final reaction volume of 60μL/tube and 2) one isotype control tube containing PE-conjugated mouseIgG to account for nonspecific primary antibody binding in a finalreaction volume of 60 μL/tube.

The tubes are gently mixed, incubated for 20 minutes at 20-24° C.followed by the addition of 400 μL of cold (2-8° C.) 1% formaldehyde andstorage in the cold, protected from light for two hours. Prior toanalyzing the fixed platelet samples, the FACS machine settings for thepeak channel, gating, axes, quadrant locations, and marker boundariesfor the appropriate regions of data acquisition are determined bycollecting a minimum of 10,00 events from a sample of freshly sonicatedlatex beads with a uniform diameter of 1 μM (CML Cat #C37483) diluted in0.9% saline. After applying these settings, a minimum of 100,000 eventsis acquired for the isotype control and for the CD61 stained sample. Thecounts obtained on the CD61 sample will register events for CD-61positive fluorescence-stained PLTs, CD-61 positive mpPLTs and for thenumber of Trucount fluorescent beads detected. Events counted in thepreset regions are analyzed in the platelet size range 2-4 microns andin the mpPLTs quadrant corrected for nonspecific binding detected in theisotype control. Data are normalized to the efficiency of Trucount beadsdetected using the following formula based on the known number ofTrucount beads per tube provided by the manufacturer.

#Events Counted/#Beads Counted X #TruCount Beads per tube/SampleVolume=mpPLT/μL or PLT/μL.

Additionally, microscopic inspection is utilized to assess hESC-PLT andhiPSC-PLT morphology. Morphology is assessed by DifferentialInterference Contrast Microscopy (DIC) and by confirming staining forβ1-tubulin in a characteristic circumferential band of microtubulesunique to platelets. Briefly, platelets are fixed in 4% formaldehyde andcentrifuged onto 1 μg/ml poly-1-lysine-coated coverslips, permeabilizedwith 0.5% Triton X-100, and blocked in immunofluorescence blockingbuffer (0.5 g BSA, 0.25 ml of 10% sodium azide, and 5 ml FCS in 50 mlPBS) for a minimum of two hours before antibody labeling. To demarcatepermeabilized cells, samples are incubated with a rabbit polyclonalprimary antibody for human β1-tubulin generated against the C-terminalpeptide sequence CKAVLEEDEEVTEEAEMEPEDKGH (Genemed Synthesis, Inc.) (SEQID NO:1). Samples are then treated with a secondary goat anti-rabbitantibody conjugated to an Alexa Fluor 568 nm (Invitrogen; MolecularProbes), with extensive washes with PBS between and after theincubations. Coverslips are mounted (Aqua Polymount; Polysciences) ontomicroscope slides. As background controls, slides are incubated with thesecondary antibody alone and images are adjusted to account fornonspecific binding of antibodies. Samples are examined with amicroscope equipped with an oil immersion objective or differentialinterference contrast objective. Images are obtained using acharge-coupled device camera. Images were analyzed using the MetaMorphimage analysis software (Molecular Devices) and ImageJ (NationalInstitutes of Health). Characteristic features of resting platelets areassessed as follows: Differential-interference contrast (DIC)microscopy: Disc-shaped, approximately 2-3 μM in diameter PI-Tubulinstaining: A prominent, circumferential band of microtubules with adiameter similar to resting platelets. See FIG. 21.

Platelets (hESC-PLTs and hiPSC-PLTs) are additionally tested to confirmthe absence of pluripotent cells (e.g., by PCR, immunofluorescence,and/or FACS to detect pluripotency markers). To enhance sensitivity,detection methods may be coupled with procedures to concentratepotential cellular contaminants by trapping with filters, matrices orgradients coupled with low speed centrifugation to pellet cells andwhile leaving platelets in the supernatant.

Platelets (hESC-PLTs and hiPSC-PLTs) are additionally tested for thepresence of microorganisms according to the immersion method, USP <21>,CFR 610.12.

Platelets (hESC-PLTs and hiPSC-PLTs) are additionally tested forendotoxin. Gram-negative bacterial endotoxins are quantified using thePyrotell® Gel Clot Endotoxin System (Associates of Cape Cod, Inc.).Appropriate negative, positive, and positive product controls areprepared. Positive product controls are inhibition controls and consistof the specimen or dilution of specimen to which standard endotoxin isadded. The samples are added directly to the Pyrotell® reagent and mixedthoroughly. The reaction tubes are incubated at 37° C. 1° C. for 60±2minutes. A positive test is indicated by the formation of a gel whichdoes not collapse when the tube is inverted. Endotoxin is quantified byfinding the endpoint in a series of specimen dilutions. In the absenceof the endotoxin series, a positive control of know concentration may beincluded with the tests. The endotoxin assay will be validated for usewith PLT samples and is performed in a manner consistent with the 1987FDA guidance on endotoxin validation.

Platelets (hESC-PLTs and hiPSC-PLTs) are additionally tested formycoplasma. After centrifugation of platelets prior to running the HSAgradient, the supernatants (e.g., consisting of conditioned StemSpan ACFmedium) are collected and pooled. Samples of purified platelets from thefinal product batch and the conditioned medium are sent for mycoplasmatesting. Mycoplasma detection is performed as per the EuropeanPharmacopoeia and US Pharmacopoeia Guidelines with indirect cultivationon indicator cell cultures and direct inoculation on agar plates andinto broth.

Example 6. Assay for Platelet Potency and PAC-1 Binding

Platelets of the disclosure may be assessed for potency and/or PAC-1binding using the methods described in this example.

Platelet activation in vivo induces conformational changes in αIIbβ3integrin activating the fibrinogen receptor function of the GPIIb/IIIacomplex leading to enhanced ligand binding. Activated platelet bind tosubstrates including fibrinogen and Von Willebrand Factor and stimulatethrombus formation at the site of vascular injury. To determine theextent of functional αIIbβ3 integrin expression that occurs uponplatelet activation, hESC-PLTs, iPSC-PLTs, and purified normal humanplatelets are activated and assessed for the extent of PAC-1 binding ascompared to resting control PLTs. The PAC-1 is a fibrinogen mimetic thatbinds exclusively to the activated conformation of the αIIbβ3 integrin.

PLTs are counted and a minimum of 100,000 PLTs is removed and dilutedwith additional medium to a density of approximately 20 PLT/uL.PAC1-FITC (BD, 1:100 dilution) and antibodies (CD41a-APC-conjugated(allophycocyanin) 1:100 and CD42b-PE-conjugated (phycoerythrin) 1:100(BD Bioscience, San Jose, Calif.) are added to the PLT sample. One halfof the sample (250 uL) is dispensed into each of two 5 mL FACS tubes. Toone tube, thrombin (Sigma) is added to a final concentration of 1U/mL.Activated PLTs exposed to thrombin and control samples are incubated atroom temperature for 15-20 minutes. Samples undergo FACS analysis withforward versus side scatter gating being determined using human bloodplatelets as controls. PAC-1 binding (activation) is quantified bycomparing the number of CD41a and PAC-1 positive events in the activatedto those in the unactivated control.

1. A pharmaceutical preparation that is suitable for use in a humanpatient comprising at least 10⁸ platelets, wherein the preparation issubstantially free of leukocytes and wherein substantially all of theplatelets are functional, or a bioreactor having weakly adherent ornon-adherent megakaryocytes that produce functional platelets withoutfeeder cells, or a composition comprising at least 10⁹ MLPs, or acryopreserved composition comprising MLPs, optionally comprisingcomprising 10⁹ to 10¹⁴ MLPs, further optionally 10⁹, 10¹⁰, 10¹¹, 10¹²,10¹³ or 10¹⁴ MLPs or a bank comprising cryopreserved MLPs. 2-13.(canceled)
 14. A method for producing platelets from megakaryocytescomprising the steps of: (A) a) providing a non-adherent culture ofmegakaryocytes; b) contacting the megakaryocytes with (i) TPO or a TPOagonist to cause the formation of proplatelets in culture, wherein theproplatelets release platelets; or (ii) hematopoietic expansion mediumand optionally (1) TPO or a TPO agonist, SCF, IL-6 and IL-9 or (2) TPOor a TPO agonist, SCF, and IL-11 to cause the formation of pro-plateletsin culture, wherein the pro-platelets release platelets; and c)isolating the platelets optionally wherein the non-adherent culture ofmegakaryocytes is feeder-free, non-adherent culture that includes a cellpopulation comprising megakaryocytes positive for CD41a and CD42bexpression, and optionally wherein at least 60% of the releasedplatelets are positive for CD41a and CD42b expression, or (B)differentiating megakaryocytes positive for CD41a and CD42b expressionin a feeder-free, non-adherent culture to cause formation in the cultureof platelets, at least 60% of which are positive for CD41a and CD42b;isolating the platelets from the culture; and concentrating theplatelets to form a pharmaceutical preparation comprising at least 10⁸platelets, wherein the preparation is substantially free of leukocytesand wherein substantially all of the platelets are functional, andwherein the step of differentiating megakaryocytes comprises contactingthe megakaryocytes with: (i) Thrombopoietin (TPO) or a TPO agonist; or(ii) a hematopoietic expansion medium, wherein the hematopoieticexpansion medium comprises TPO or a TPO agonist and one or more of StemCell Factor (SCF), Interleukin (IL)-6, IL-9, and IL-11. 15-38.(canceled)
 39. A pharmaceutical preparation comprising plateletsproduced by the method of claim
 14. 40-41. (canceled)
 42. Use of thecomposition of claim 1 in the manufacture of a medicament for thetreatment of a patient in need thereof or suffering from a disease ordisorder affecting clotting or a disease or disorder treatable thereby,or a method of treating a patient in need of platelet transfusion,comprising administering a composition of claim 1 to said patient.43-45. (canceled)
 46. A method for producing hemogenic endothelial(PVE-HE) cells comprising differentiating pluripotent stem cells invitro, thereby forming a cell population comprising hemogenicendothelial cells, wherein the pluripotent stem cells are differentiatedwithout embryoid body formation.
 47. The method of claim 46, wherein thepluripotent stem cells are differentiated in the presence of one or moreextracellular matrix components.
 48. The method of claim 47, wherein theone or more extracellular matrix components is collagen, optionallycollagen IV.
 49. The method of claim 46, wherein the pluripotent stemcells are differentiated into hemogenic endothelial cells by culturingthe pluripotent stem cells under feeder-free conditions in adifferentiation induction medium (DIM) comprising BMP4, bFGF and VEGF,until CD31+ hemogenic endothelial cells are formed.
 50. The method ofclaim 49, wherein the pluripotent stem cells are first cultured on acollagen-coated surface in a culture medium comprising a ROCK inhibitor,and then cultured in the DIM.
 51. The method of claim 46, wherein thepluripotent stem cells are differentiated into hemogenic endothelialcells by (i) culturing the pluripotent stem cells under feeder-freeconditions on a collagen IV coated surface in the presence of a culturemedium comprising 10 μM ROCK inhibitor Y27632, (ii) replacing theculture medium of (i) with a differentiation induction medium (DIM)comprising BMP4, bFGF and VEGF, and culturing until CD31+ hemogenicendothelial cells are formed.
 52. The method of claim 46, wherein thepluripotent stem cells are differentiated into hemogenic endothelialcells under low oxygen conditions comprising 1% to 10% oxygen, 2% to 8%oxygen, 3% to 7% oxygen, 4% to 6% oxygen, or about 5% oxygen.
 53. Themethod of claim 46, wherein the pluripotent stem cells aredifferentiated into hemogenic endothelial cells at about 5% oxygen. 54.The method of claim 46, wherein the pluripotent stem cells are inducedpluripotent stem cells (iPSCs).
 55. The method of claim 46, wherein thepluripotent stem cells are embryonic stem cells (ES cells).
 56. Themethod of claim 46, wherein the hemogenic endothelial cells expressCD31/PECAM1.
 57. The method of claim 46, wherein the hemogenicendothelial cells express CD31/PECAM1, CD144/VE-Cad, and CD105/endoglin.58. The method of claim 46, wherein at least 70% of cells in the cellpopulation express CD31/PECAM1, CD144/VE-Cad, and CD105/endoglin. 59.The method of claim 46, wherein the hemogenic endothelial cells expressCD34, CD309/KDR, CD146, and/or CD184/CXCR4.
 60. The method of claim 46,further comprising cryopreserving the cell population comprising thehemogenic endothelial cells.
 61. A composition comprising a cellpopulation, wherein at least 70% of cells in the population areCD31/PECAM1+, CD144/VE-Cad+, and CD105/endoglin+hemogenic endothelialcells, wherein the hemogenic endothelial cells are produced by in vitrodifferentiation of pluripotent stem cells, optionally wherein at least80% of cells in the population are CD31/PECAM1+, CD144/VE-Cad+, andCD105/endoglin+hemogenic endothelial cells, further optionally whereinthe cells in the population are determined to be CD31/PECAM1+,CD144/VE-Cad+, and CD105/endoglin+ by immunofluorescence, furtheroptionally wherein the CD31/PECAM1+, CD144/VE-Cad+, andCD105/endoglin+hemogenic endothelial cells express CD34, CD309/KDR,CD146, and/or CD184/CXCR4, further optionally wherein the composition iscryopreserved.